Mutation of the ear motif of class II HD-Zip polypeptides

ABSTRACT

The application describes producing polynucleotide variants of the AtHB 17 clade members and introducing the mutant variants into plants to improve plant traits. The mutant polynucleotides encode polypeptides that comprise mutations in the EAR motifs.

This application is a 371 National Stage application of InternationalApplication No. PCT/US13/35640, filed Apr. 8, 2013, which claims thepriority of U.S. Provisional Patent Application No. 61/621,980, filedApr. 9, 2012, all of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present disclosure relates to plant genomics and plant improvement,increasing a plant's photosynthesis and the yield.

BACKGROUND

Yield is one of the most important agronomic traits and is determined bythe interaction of specific genetics within the crops with environmentalfactors. An important approach to increasing yield potential isincreasing overall plant productivity through increasing photosyntheticefficiency or resource utilization.

The Arabidopsis sequence AtHB17 (At2g01430, or G1543) encodes a memberof the class II sub-group of the homeodomain-leucine zipper protein(HD-Zip) family of transcription factors. It triggers increases inchlorophyll levels when overexpressed in plants (U.S. Pat. No.7,511,190).

The instant disclosure provides methods for altering the proteinstructure of AtHB17 and other class II HD-ZIP closely-related to AtHB17,i.e. introducing mutations to the EAR motif of the AtHB17 cladesequences and overexpressing those EAR mutation variants in plants toconfer beneficial phenotypes.

SUMMARY

The instant disclosure provides a method to reduce or eliminate certainphenotypes that may be associated with the overexpression of HD-ZIPpolypeptides while maintaining the high rates of photosynthesis that canbe associated with said overexpression. Certain phenotypes may bereduced or eliminated by introducing an EAR mutation variant of AtHB17or related class II HD-ZIP polypeptides (AtHB17 clade members) into atarget plant. Typically, the conserved EAR motif SEQ ID NO: 50 in theAtHB17 clade members is replaced with the mutant EAR motif, SEQ ID NO: 1(i.e., at least one of the conserved leucines within the EAR motifs isreplaced with an amino acid other than leucine, valine and isoleucine).These EAR mutant variants thus do not comprise SEQ ID NO: 50. Thesevariants can regulate stomatal aperture of the transgenic plants in amanner similar to wild-type plants, yet retains increased chlorophylllevels and enhanced photosynthetic capacity. The combination of theseproperties results in increased photosynthetic rates in transgenicplants overexpressing the EAR mutation variants.

The instant disclosure provides a transgenic C3 or C4 plant thatcomprises a polynucleotide encoding a EAR mutation variant thatcomprises a mutant EAR motif of X₁-X₂-X₃-X₄-X₅ (set forth in SEQ ID NO:1), where X₁ and X₃ are any amino acid other than leucine, isoleucine orvaline; X₂ and X₄ are any amino acid; and X₅ is isoleucine, leucine ormethionine. The polypeptide additionally comprises a homeodomain thatshares an amino acid percentage identity to the homeodomain of any ofSEQ ID NO: 2-20; and a homeobox-associated leucine zipper (HALZ) domainthat shares an amino acid percentage identity to the HALZ domain of anyof SEQ ID NO: 2-20. The amino acid percentage identity is selected fromthe group consisting of at least 60%, at least 61%, at least 62%, atleast 63%, at least 64%, at least 65%, at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, and about 100%.

The transgenic C₃ or C4 plant overexpressing said EAR mutation varianthas higher nitrogen use efficiency, lower chlorophyll levels, greaterstomatal conductance, greater size, greater biomass, greater growthrate, and/or greater yield relative to a first control plant of the samespecies that comprises a recombinant polynucleotide encoding any of SEQID NO: 2-20.

In some embodiments of the disclosure, the transgenic C3 or C4 plantsalso have greater nitrogen use efficiency, greater size, greaterbiomass, greater yield, greater growth rate, greater chlorophyll levels,greater photosynthetic capacity, greater photosynthetic rate, and/orgreater stomatal conductance, relative to a second control plant that isa wild type or non-transformed plant of the same species.

In some embodiments of the instant disclosure, the polypeptide is atleast 30%, at least 31%, at least 32%, at least 33%, at least 34%, atleast 35%, at least 36%, at least 37%, at least 38%, at least 39%, atleast 40%, at least 41%, at least 42%, at least 43%, at least 44%, atleast 45%, at least 46%, at least 47%, at least 48%, at least 49%, atleast 50%, at least 51%, at least 52%, at least 53%, at least 54%, atleast 55%, at least 56%, at least 57%, at least 58%, at least 59%, atleast 60%, at least 61%, at least 62%, at least 63%, at least 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, atleast 70%, at least 71%, at least 72%, at least 73%, at least 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, atleast 80%, at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orabout 100% identical to the full length sequence of SEQ ID NO: 2-20.

In a preferred embodiment, X₁ or X₃, or both X₁ and X₃, are alanine.

In another preferred embodiment, the EAR mutant motif is A-D-A-T-I (SEQID NO: 89).

The instant disclosure also provides a method for conferring to a C3 orC4 plant greater nitrogen use efficiency, greater chlorophyll levels,greater photosynthetic capacity, greater photosynthetic rate, greaterstomatal conductance, greater size, greater biomass, or greater yieldrelative to a wild type or non-transformed plant of the same species ora plant transformed with an empty vector. The method comprises the stepsof:

-   -   (a) providing a polynucleotide encoding a first polypeptide that        comprises        -   (1) a consensus EAR motif set forth as SEQ ID NO: 50;        -   (2) a homeodomain that shares an amino acid percentage            identity to a homeodomain of any of SEQ ID NO: 2-20; and        -   (3) a homeobox-associated leucine zipper (HALZ) domain that            shares an amino acid percentage identity to a HALZ domain            set forth in any of SEQ ID NO: 2-20;            -   wherein the amino acid percentage identity is selected                from the group consisting of: at least 60%, at least                61%, at least 62%, at least 63%, at least 64%, at least                65%, at least 66%, at least 67%, at least 68%, at least                69%, at least 70%, at least 71%, at least 72%, at least                73%, at least 74%, at least 75%, at least 76%, at least                77%, at least 78%, at least 79%, at least 80%, at least                81%, at least 82%, at least 83%, at least 84%, at least                85%, at least 86%, at least 87%, at least 88%, at least                89%, at least 90%, at least 91%, at least 92%, at least                93%, at least 94%, at least 95%, at least 96%, at least                97%, at least 98%, at least 99%, and about 100%;    -   (b) mutagenizing the polynucleotide, wherein the mutagenized        polynucleotide encodes a EAR mutant polypeptide comprising a        mutant EAR motif of X₁-X₂-X₃-X₄-X₅ (set forth in SEQ ID NO: 1),        where X₁ and X₃ are any amino acid other than leucine,        isoleucine or valine; and X₅ is isoleucine, leucine or        methionine;    -   (c) introducing the mutagenized polynucleotide into a target        plant to produce a transgenic C3 or C4 plant, wherein expression        of the mutagenized polynucleotide confers the improved trait in        the transgenic C3 or C4 plant; and    -   (d) optionally, selecting the transgenic C3 or C4 plant having        greater nitrogen use efficiency, greater chlorophyll levels,        greater photosynthetic capacity, greater photosynthetic rate,        greater stomatal conductance, greater size, greater biomass, or        greater yield relative to a wild type or non-transformed plant        of the same species.

In another embodiment of the instant disclosure, said EAR mutantpolypeptide has decreased repression activity as compared to the firstHD-ZIP polypeptide which comprises the native EAR motif (SEQ ID NO: 1).

In a preferred embodiment, the first polypeptide is a sequence selectedfrom SEQ ID NO: 2-20.

In another preferred embodiment of the instant disclosure, thetransgenic plant that is generated by transforming the EAR mutantpolynucleotide is a soybean or a corn plant.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING AND DRAWINGS

The Sequence Listing provides exemplary polynucleotide and polypeptidesequences of the instant disclosure. The traits associated with the useof the sequences are included in the Examples. The sequence listing wascreated on Apr. 5, 2013 and is 101,575 bytes (99.1 kilobytes as measuredin MS Windows). The entire content of the sequence listing is herebyincorporated by reference.

FIGS. 1A-1G show the alignment of the AtHB17 subclade sequences. Theboxes indicate the conserved EAR motifs, the homeodomains (HD), thehomeodomain-associated leucine zippers (HALZ), and the “CPSCE” domain.In each column, asterisks indicate identical amino acid residues, colonsindicate conservative substitutions and periods indicate similar aminoacids. SEQ ID NOs: of the respective proteins are shown in parentheses.

FIGS. 2A and 2B show the response of light-saturated photosynthesis(A_(sat)) to leaf sub-stomatal CO₂ concentration (C_(i)) for two35S::AtHB17 native protein lines (FIG. 2A) and two 35S::AtHB17 lineswith a mutation in the EAR domain (AtHB17 L84A L86A) (FIG. 2B). All dataare plotted against an empty vector control line to which a polynomialregression has been fitted. All data points are the mean±1 standarderror of measurements made on leaves of at least six replicate plants.

Legend for FIG. 2A and FIG. 2B:

◯ represents pMEN65 lines

Δ represents 35S::AtHB17 373-22 lines

▴ represents 35S::AtHB17 378-6 lines

□ represents EAR Z193359-1 lines

▪ represents EAR Z193358-14 lines

FIGS. 3A and 3B show the stomatal conductance (g_(s)) measured onmultiple 35S::AtHB17 native protein lines (FIG. 3A) and two 35S::AtHB17EAR mutant lines (AtHB17 L84A L86A) (FIG. 3B) along with empty vectorcontrols (pMEN65) in several independent experiments. For eachindependent experiment, the empty vector control line (solid bar) isplotted on the left of the 35S::AtHB17 or 35S::AtHB17 variant line(s)measured in the same experiment (white bars). All measurements were madeat an atmospheric CO₂ concentration of 400 μmol mol⁻¹ after acclimationto a light condition that is saturating for photosynthesis (thephotosynthetic photon flux is at least 700 (μmol m⁻² s⁻¹)). All datashown are the mean±1 standard error of measurements made on at leastfive replicate plants. An asterisk above a 35S::AtHB17 or AtHB17 variantbar indicates that the mean shown for that line is significantlydifferent than the control measured at the same time (solid bar onleft).

FIG. 4 shows light-saturated photosynthesis (A_(sat)) measured onmultiple 35S::AtHB17 lines (FIG. 4A) and two 35S::AtHB17 EAR mutantlines (AtHB17 L84A L86A) (FIG. 4B) along with empty vector controls(pMEN65) for data collected after acclimation to a current atmosphereduring several independent experiments. For each experiment the emptyvector control line (solid bar) is plotted on the left of the35S::AtHB17 lines measured in the same experiment (white bars). Allmeasurements were made as described in FIG. 3. All data shown are themean±1 standard error of measurements made on at least five replicateplants. * positioned above a 35S::AtHB17 bar indicates that the meanshown for that line is significantly different from the control measuredat the same time (solid bar on left).

FIG. 5 shows the percentage change in stomatal conductance andlight-saturated photosynthesis plotted for 35S::AtHB17 lines expressingeither the native protein, or the protein with a mutation in the EARdomain, relative to a control line. Data presented is the mean andstandard error of the percent effect size determined for up to fourindependent lines in at least six independent experiments for eachprotein variant.

FIG. 6 shows trend plots of the expression of several genes involved inthe regulation of stomatal movement, from a transcript profilingexperiment comparing plants overexpressing AtHB17 (35S::AtHB17,represented in each plot by the “1” on each x-axis) and plantsoverexpressing an AtHB17 variant in which the EAR motif was mutated(35S::AtHB17 L84A_L86A, represented in each plot by the “2” on eachx-axis) to relative to control plants. Data points are the log 2 of theratio of intensity of a given probeset in the experimental plants vs.the controls. In FIG. 6A, the solid line represents expression ofAtBGL1. In FIG. 6B, the solid line represents expression of KAT1 and thedashed line represents expression of KAT2. In FIG. 6C, the solid linerepresents expression of ABI1 and the dashed line represents expressionof RD20. In FIG. 6D, the solid line represents expression of CPK6 andthe dashed line represents expression MYB60.

FIGS. 7A and 7B show chlorophyll content as measured by a Minolta SPADmeter and morphological phenotypes of the Arabidopsis plantsoverexpressing the EAR mutant and the native AtHB17 polypeptide. FIG. 7Ashows the chlorophyll levels (SPAD) of plants from an empty vector line(“35S::MCS”), a wild type line (“Col_Wt”), a transgenic lineoverexpressing the native AtHB17 polypeptide (“35S::AtHB17”) and fourlines of plants overexpressing EAR mutation variant (“Z193359-1”,“Z193358-14”, “Z193363-4”, and “Z193346-14”), “35S::AtHB17 (L84A_L86A)”.The EAR mutant overexpressors were larger in size and biomass comparedto empty vector lines and the wild type plants, and significantly largerand had more biomass than AtHB17 overexpressors, as indicated FIG. 7B.

FIG. 8 shows the results of a protoplast-based transcriptional assayanalyzing the effect of the EAR mutation on transcriptional repression.Chloramphenicol acetyltransferase (CAT), AtHB17, and the EAR mutant(AtHB17 L84A L86A) constructs were co-transfected into Arabidopsismesophyll protoplasts with a prHAT1::GUS reporter gene. Data are meanfluorescence reading measuring GUS-mediated substrate of threereplicates.

FIG. 9 shows that the EAR mutation did not impair the DNA bindingactivity of AtHB17. An AtHB17Δ1-73 deletion polypeptide which lacks theamino acid residues 1-73 (“Δ1-73”), an AtHB17Δ1-73 L84A L86A mutantpolypeptide containing an EAR mutation in addition to a deletion ofamino acid residues 1-73 (“Δ1-73 L84A L86A”) were analyzed using anELISA-based binding assay as described in the Examples. The mutantpolypeptide (“Δ1-73 L84A L86A”) showed higher DNA-binding activity ascompared to the deletion polypeptide (“Δ1-73”). The solid bars showresults obtained in AtHB17 activity assays with the unmutated HD-ZIPClass II binding site (SEQ ID NO: 48), and the white bars representresults with the mutated HD-ZIP Class II binding site (SEQ ID NO: 49).

DETAILED DESCRIPTION

The present disclosure relates to polynucleotides and polypeptides formodifying phenotypes of plants, particularly those associated withincreased abiotic stress tolerance and increased yield with respect to acontrol plant (for example, a wild-type plant, a non-transformed plant,or a plant transformed with an “empty” nucleic acid construct or vectorlacking a polynucleotide of interest comprised within a nucleic acidconstruct introduced into a plant). Throughout this disclosure, variousinformation sources are referred to and/or are specificallyincorporated. The information sources include scientific journalarticles, patent documents, textbooks, and World Wide Webbrowser-inactive page addresses. While the reference to theseinformation sources clearly indicates that they can be used by one ofskill in the art, each and every one of the information sources citedherein are specifically incorporated in their entirety, whether or not aspecific mention of “incorporation by reference” is noted. The contentsand teachings of each and every one of the information sources can berelied on and used to make and use embodiments of the instantdisclosure.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include the plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “a host cell”includes a plurality of such host cells, and a reference to “a stress”is a reference to one or more stresses and equivalents thereof known tothose skilled in the art, and so forth.

Definitions

“Polynucleotide” is a nucleic acid molecule comprising a plurality ofpolymerized nucleotides, e.g., at least 15 consecutive polymerizednucleotides. A polynucleotide may be a nucleic acid, oligonucleotide,nucleotide, or any fragment thereof. In many instances, a polynucleotidecomprises a nucleotide sequence encoding a polypeptide (or protein) or adomain or fragment thereof. Additionally, the polynucleotide maycomprise a promoter, an intron, an enhancer region, a polyadenylationsite, a translation initiation site, 5′ or 3′ untranslated regions, areporter gene, a selectable marker, or the like. The polynucleotide canbe single-stranded or double-stranded DNA or RNA. The polynucleotideoptionally comprises modified bases or a modified backbone. Thepolynucleotide can be, e.g., genomic DNA or RNA, a transcript (such asan mRNA), a cDNA, a PCR product, a cloned DNA, a synthetic DNA or RNA,or the like. The polynucleotide can be combined with carbohydrate,lipids, protein, or other materials to perform a particular activitysuch as transformation or form a useful composition such as a peptidenucleic acid (PNA). The polynucleotide can comprise a sequence in eithersense or antisense orientations. “Oligonucleotide” is substantiallyequivalent to the terms amplimer, primer, oligomer, element, target, andprobe and is preferably single-stranded.

A “recombinant polynucleotide” is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into anucleic acid construct, or otherwise recombined with one or moreadditional nucleic acid.

An “isolated polynucleotide” is a polynucleotide, whether naturallyoccurring or recombinant, that is present outside the cell in which itis typically found in nature, whether purified or not. Optionally, anisolated polynucleotide is subject to one or more enrichment orpurification procedures, e.g., cell lysis, extraction, centrifugation,precipitation, or the like.

“Gene” or “gene sequence” refers to the partial or complete codingsequence of a gene, its complement, and its 5′ or 3′ untranslatedregions. A gene is also a functional unit of inheritance, and inphysical terms is a particular segment or sequence of nucleotides alonga molecule of DNA (or RNA, in the case of RNA viruses) involved inproducing a polypeptide chain. The latter may be subjected to subsequentprocessing such as chemical modification or folding to obtain afunctional protein or polypeptide. A gene may be isolated, partiallyisolated, or found with an organism's genome. By way of example, atranscription factor gene encodes a transcription factor polypeptide,which may be functional or require processing to function as aninitiator of transcription.

Operationally, genes may be defined by the cis-trans test, a genetictest that determines whether two mutations occur in the same gene andthat may be used to determine the limits of the genetically active unit(Rieger et al., 1976). A gene generally includes regions preceding(“leaders”; upstream) and following (“trailers”; downstream) the codingregion. A gene may also include intervening, non-coding sequences,referred to as “introns”, located between individual coding segments,referred to as “exons”. Most genes have an associated promoter region, aregulatory sequence 5′ of the transcription initiation codon (there aresome genes that do not have an identifiable promoter). The function of agene may also be regulated by enhancers, operators, and other regulatoryelements.

A “polypeptide” is an amino acid sequence comprising a plurality ofconsecutive polymerized amino acid residues e.g., at least 15consecutive polymerized amino acid residues. In many instances, apolypeptide comprises a polymerized amino acid residue sequence that isa transcription factor or a domain or portion or fragment thereof.Additionally, the polypeptide may comprise: (i) a localization domain;(ii) an activation domain; (iii) a repression domain; (iv) anoligomerization domain; (v) a protein-protein interaction domain; (vi) aDNA-binding domain; or the like. The polypeptide optionally comprisesmodified amino acid residues, naturally occurring amino acid residuesnot encoded by a codon, non-naturally occurring amino acid residues.

“Protein” refers to an amino acid sequence, oligopeptide, peptide,polypeptide or portions thereof whether naturally occurring orsynthetic.

A “recombinant polypeptide” is a polypeptide produced by translation ofa recombinant polynucleotide. A “synthetic polypeptide” is a polypeptidecreated by consecutive polymerization of isolated amino acid residuesusing methods well known in the art. An “isolated polypeptide,” whethera naturally occurring or a recombinant polypeptide, is more enriched in(or out of) a cell than the polypeptide in its natural state in awild-type cell, e.g., more than about 5% enriched, more than about 10%enriched, or more than about 20%, or more than about 50%, or more,enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,enriched relative to wild type standardized at 100%. Such enrichment isnot the result of a natural response of a wild-type plant.Alternatively, or additionally, the isolated polypeptide is separatedfrom other cellular components with which it is typically associated,e.g., by any of the various protein purification methods herein.

“Homology” refers to sequence similarity between a reference sequenceand at least a fragment of a newly sequenced clone insert or its encodedamino acid sequence.

“Identity” or “similarity” refers to sequence similarity between twopolynucleotide sequences or between two polypeptide sequences, withidentity being a more strict comparison. The phrases “percent identity”and “% identity” refer to the percentage of sequence similarity found ina comparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar, or any integer value therebetween.Identity or similarity can be determined by comparing a position in eachsequence that may be aligned for purposes of comparison. When a positionin the compared sequence is occupied by the same nucleotide base oramino acid, then the molecules are identical at that position. A degreeof similarity or identity between polynucleotide sequences is a functionof the number of identical, matching or corresponding nucleotides atpositions shared by the polynucleotide sequences. A degree of identityof polypeptide sequences is a function of the number of identical aminoacids at corresponding positions shared by the polypeptide sequences. Adegree of homology or similarity of polypeptide sequences is a functionof the number of amino acids at corresponding positions shared by thepolypeptide sequences.

“Alignment” refers to a number of nucleotide bases or amino acid residuesequences aligned by lengthwise comparison so that components in common(i.e., nucleotide bases or amino acid residues at correspondingpositions) may be visually and readily identified. The fraction orpercentage of components in common is related to the homology oridentity between the sequences. Alignments such as those of FIGS. 1A-1Gmay be used to identify conserved domains and relatedness within thesedomains. An alignment may suitably be determined by means of computerprograms known in the art, such as MACVECTOR software (1999) (Accelrys,Inc., San Diego, Calif.), and the European Molecular Biology OpenSoftware Suite (EMBOSS) Needle program (Rice, P., et al. 2000)

A “conserved domain” or “conserved region” as used herein refers to aregion within heterogeneous polynucleotide or polypeptide sequenceswhere there is a relatively high degree of sequence identity or homologybetween the distinct sequences. With respect to polynucleotides encodingpresently disclosed polypeptides, a conserved domain is preferably atleast nine base pairs (bp) in length. Protein sequences, includingtranscription factor sequences, that possess or encode for conserveddomains that have a minimum percentage identity and have comparablebiological activity to the present polypeptide sequences, thus beingmembers of the same clade of transcription factor polypeptides, areencompassed by the instant disclosure. Reduced or eliminated expressionof a polypeptide that comprises, for example, a conserved domain havingDNA-binding, activation or nuclear localization activity, results in thetransformed plant having similar improved traits as other transformedplants having reduced or eliminated expression of other members of thesame clade of transcription factor polypeptides.

A fragment or domain can be referred to as outside a conserved domain,outside a consensus sequence, or outside a consensus DNA-binding sitethat is known to exist or that exists for a particular polypeptideclass, family, or sub-family. In this case, the fragment or domain willnot include the exact amino acids of a consensus sequence or consensusDNA-binding site of a transcription factor class, family or sub-family,or the exact amino acids of a particular transcription factor consensussequence or consensus DNA-binding site. Furthermore, a particularfragment, region, or domain of a polypeptide, or a polynucleotideencoding a polypeptide, can be “outside a conserved domain” if all theamino acids of the fragment, region, or domain fall outside of a definedconserved domain(s) for a polypeptide or protein. Sequences havinglesser degrees of identity but comparable biological activity areconsidered to be equivalents.

As one of ordinary skill in the art recognizes, conserved domains may beidentified as regions or domains of identity to a specific consensussequence (see, for example, Riechmann et al., 2000a, 2000b). Thus, byusing alignment methods well known in the art, the conserved domains ofthe plant polypeptides may be determined.

The conserved domains for many of the polypeptide sequences of thepresent disclosure are listed in Table 1 and are indicated by amino acidcoordinate start and stop sites. A comparison of the regions of thesepolypeptides allows one of skill in the art (see, for example, Reevesand Nissen, 1995) to identify domains or conserved domains for any ofthe polypeptides listed or referred to in this disclosure.

“Complementary” refers to the natural hydrogen bonding by base pairingbetween purines and pyrimidines. For example, the sequence A-C-G-T(5′->3′) forms hydrogen bonds with its complements A-C-G-T (5′->3′) orA-C-G-U (5′->3′). Two single-stranded molecules may be consideredpartially complementary, if only some of the nucleotides bond, or“completely complementary” if all of the nucleotides bond. The degree ofcomplementarity between nucleic acid strands affects the efficiency andstrength of hybridization and amplification reactions. “Fullycomplementary” refers to the case where bonding occurs between everybase pair and its complement in a pair of sequences, and the twosequences have the same number of nucleotides.

The terms “paralog” and “ortholog” are defined below in the sectionentitled “Orthologs and Paralogs”. In brief, orthologs and paralogs areevolutionarily related genes that have similar sequences and functions.Orthologs are structurally related genes in different species that arederived by a speciation event. Paralogs are structurally related geneswithin a single species that are derived by a duplication event.

With regard to polynucleotide variants, differences between presentlydisclosed polynucleotides and polynucleotide variants are limited sothat the nucleotide sequences of the former and the latter are closelysimilar overall and, in many regions, identical. Due to the degeneracyof the genetic code, differences between the former and latternucleotide sequences may be silent (i.e., the amino acids encoded by thepolynucleotide are the same, and the variant polynucleotide sequenceencodes the same amino acid sequence as the presently disclosedpolynucleotide. Variant nucleotide sequences may encode different aminoacid sequences, in which case such nucleotide differences will result inamino acid substitutions, additions, deletions, insertions, truncationsor fusions with respect to the similar disclosed polynucleotidesequences. These variations may result in polynucleotide variantsencoding polypeptides that share at least one functional characteristic.The degeneracy of the genetic code also dictates that many differentvariant polynucleotides can encode identical and/or substantiallysimilar polypeptides in addition to those sequences illustrated in theSequence Listing.

Also within the scope of the instant disclosure is a variant of anucleic acid listed in the Sequence Listing, that is, one having asequence that differs from the one of the polynucleotide sequences inthe Sequence Listing, or a complementary sequence, that encodes afunctionally equivalent polypeptide (i.e., a polypeptide having somedegree of equivalent or similar biological activity) but differs insequence from the sequence in the Sequence Listing, due to degeneracy inthe genetic code. Included within this definition are polymorphisms thatmay or may not be readily detectable using a particular oligonucleotideprobe of the polynucleotide encoding polypeptide, and improper orunexpected hybridization to allelic variants, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingpolypeptide.

“Allelic variant” or “polynucleotide allelic variant” refers to any oftwo or more alternative forms of a gene occupying the same chromosomallocus. Allelic variation arises naturally through mutation, and mayresult in phenotypic polymorphism within populations. Gene mutations maybe “silent” or may encode polypeptides having altered amino acidsequence. “Allelic variant” and “polypeptide allelic variant” may alsobe used with respect to polypeptides, and in this case the terms referto a polypeptide encoded by an allelic variant of a gene.

As used herein, “polynucleotide variants” may also refer topolynucleotide sequences that encode paralogs and orthologs of thepresently disclosed polypeptide sequences. “Polypeptide variants” mayrefer to polypeptide sequences that are paralogs and orthologs of thepresently disclosed polypeptide sequences.

Differences between presently disclosed polypeptides and polypeptidevariants are limited so that the sequences of the former and the latterare closely similar overall and, in many regions, identical. Presentlydisclosed polypeptide sequences and similar polypeptide variants maydiffer in amino acid sequence by one or more substitutions, additions,deletions, fusions and truncations, which may be present in anycombination. These differences may produce silent changes and result ina functionally equivalent polypeptide. Thus, it will be readilyappreciated by those of skill in the art, that any of a variety ofpolynucleotide sequences is capable of encoding the polypeptides andhomolog polypeptides of the instant disclosure. A polypeptide sequencevariant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties. Deliberate aminoacid substitutions may thus be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as a significant amount ofthe functional or biological activity of the polypeptide is retained.For example, negatively charged amino acids may include aspartic acidand glutamic acid, positively charged amino acids may include lysine andarginine, and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; and phenylalanine and tyrosine. More rarely, a variant mayhave “non-conservative” changes, e.g., replacement of a glycine with atryptophan. Similar minor variations may also include amino aciddeletions or insertions, or both. Related polypeptides may comprise, forexample, additions and/or deletions of one or more N-linked or O-linkedglycosylation sites, or an addition and/or a deletion of one or morecysteine residues. Guidance in determining which and how many amino acidresidues may be substituted, inserted or deleted without abolishingfunctional or biological activity may be found using computer programswell known in the art, for example, DNASTAR software (see U.S. Pat. No.5,840,544).

“Fragment”, with respect to a polynucleotide, refers to a clone or anypart of a polynucleotide molecule that retains a usable, functionalcharacteristic. Useful fragments include oligonucleotides andpolynucleotides that may be used in hybridization or amplificationtechnologies or in the regulation of replication, transcription ortranslation. A “polynucleotide fragment” refers to any subsequence of apolynucleotide, typically, of at least 9 consecutive nucleotides,preferably at least 30 nucleotides, more preferably at least 50nucleotides, of any of the sequences provided herein. Exemplarypolynucleotide fragments are the first sixty consecutive nucleotides ofthe polynucleotides listed in the Sequence Listing. Exemplary fragmentsalso include fragments that comprise a region that encodes a conserveddomain of a polypeptide. Exemplary fragments also include fragments thatcomprise a conserved domain of a polypeptide.

Fragments may also include subsequences of polypeptides and proteinmolecules, or a subsequence of the polypeptide. Fragments may have usesin that they may have antigenic potential. In some cases, the fragmentor domain is a subsequence of the polypeptide which performs at leastone biological function of the intact polypeptide in substantially thesame manner, or to a similar extent, as does the intact polypeptide. Forexample, a polypeptide fragment can comprise a recognizable structuralmotif or functional domain such as a DNA-binding site or domain thatbinds to a DNA promoter region, an activation domain, or a domain forprotein-protein interactions, and may initiate transcription. Fragmentscan vary in size from as few as 3 amino acid residues to the full lengthof the intact polypeptide, but are preferably at least 30 amino acidresidues in length and more preferably at least 60 amino acid residuesin length.

The instant disclosure also encompasses production of DNA sequences thatencode polypeptides and derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available nucleic acid constructs and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingpolypeptides or any fragment thereof.

The term “plant” includes whole plants, shoot vegetativeorgans/structures (for example, leaves, stems and tubers), roots,flowers and floral organs/structures (for example, bracts, sepals,petals, stamens, carpels, anthers and ovules), seed (including embryo,endosperm, and seed coat) and fruit (the mature ovary), plant tissue(for example, vascular tissue, ground tissue, and the like) and cells(for example, guard cells, egg cells, epidermal cells, mesophyll cells,protoplasts, and the like), and progeny of same. The class of plantsthat can be used in the method of the instant disclosure is generally asbroad as the class of higher and lower plants amenable to transformationtechniques, including angiosperms (monocotyledonous and dicotyledonousplants), gymnosperms, ferns, horsetails, psilophytes, lycophytes,bryophytes, and multicellular algae (Ku et al., 2000; and see alsoTudge, 2000).

A “control plant” as used in the present disclosure refers to a plantcell, seed, plant component, plant tissue, plant organ or whole plantused to compare against transformed, transgenic or genetically modifiedplant for the purpose of identifying an enhanced phenotype in thetransformed, transgenic or genetically modified plant. A control plantmay in some cases be a transformed or transgenic plant line thatcomprises an empty nucleic acid construct or marker gene, but does notcontain the recombinant polynucleotide of the present disclosure that isexpressed in the transformed, transgenic or genetically modified plantbeing evaluated. In general, a control plant is a plant of the same lineor variety as the transformed, transgenic or genetically modified plantbeing tested. A suitable control plant would include a geneticallyunaltered or non-transgenic plant of the parental line used to generatea transformed or transgenic plant herein.

“Wild type” or “wild-type”, as used herein, refers to a plant cell,seed, plant component, plant tissue, plant organ or whole plant that hasnot been genetically modified or treated in an experimental sense.Wild-type cells, seed, components, tissue, organs or whole plants may beused as controls to compare levels of expression and the extent andnature of trait modification with cells, tissue or plants of the samespecies in which a polypeptide's expression is altered, e.g., in that ithas been knocked out, overexpressed, or ectopically expressed.

“Genetically modified” refers to a plant or plant cell that has beenmanipulated through, for example, transformation (as defined below) ortraditional breeding methods involving crossing, genetic segregation,selection, and/or mutagenesis approaches to obtain a genotype exhibitinga trait modification of interest.

“Transformation” refers to the transfer of a foreign polynucleotidesequence into the genome of a host organism such as that of a plant orplant cell. Typically, the foreign genetic material has been introducedinto the plant by human manipulation, but any method can be used as oneof skill in the art recognizes. Examples of methods of planttransformation include Agrobacterium-mediated transformation (De Blaereet al., 1987) and biolistic methodology (U.S. Pat. No. 4,945,050 toKlein et al.).

A “transformed plant”, which may also be referred to as a “transgenicplant” or “transformant”, generally refers to a plant, a plant cell,plant tissue, seed or calli that has been through, or is derived from aplant cell that has been through, a stable or transient transformationprocess in which a “nucleic acid construct” that contains at least oneexogenous polynucleotide sequence is introduced into the plant. The“nucleic acid construct” contains genetic material that is not found ina wild-type plant of the same species, variety or cultivar, or maycontain extra copies of a native sequence under the control of itsnative promoter. The genetic material may include a regulatory element,a transgene (for example, a transcription factor sequence), a transgeneoverexpressing a protein of interest, an insertional mutagenesis event(such as by transposon or T-DNA insertional mutagenesis), an activationtagging sequence, a mutated sequence, an antisense transgene sequence, aconstruct containing inverted repeat sequences derived from a gene ofinterest to induce RNA interference, or a nucleic acid sequence designedto produce a homologous recombination event or DNA-repair based change,or a sequence modified by chimeraplasty. In some embodiments theregulatory and transcription factor sequence may be derived from thehost plant, but by their incorporation into a nucleic acid construct,represent an arrangement of the polynucleotide sequences not found in awild-type plant of the same species, variety or cultivar.

An “untransformed plant” is a plant that has not been through thetransformation process. A “stably transformed” plant, plant cell orplant tissue has generally been selected and regenerated on a selectionmedia following transformation.

A “nucleic acid construct” may comprise a polypeptide-encoding sequenceoperably linked (i.e., under regulatory control of) to appropriateinducible or constitutive regulatory sequences that allow for thecontrolled expression of polypeptide. The expression vector or cassettecan be introduced into a plant by transformation or by breeding aftertransformation of a parent plant. A plant refers to a whole plant aswell as to a plant part, such as seed, fruit, leaf, or root, planttissue, plant cells or any other plant material, e.g., a plant explant,to produce a recombinant plant (for example, a recombinant plant cellcomprising the nucleic acid construct) as well as to progeny thereof,and to in vitro systems that mimic biochemical or cellular components orprocesses in a cell.

A “trait” refers to a physiological, morphological, biochemical, orphysical characteristic of a plant or particular plant material or cell.In some instances, this characteristic is visible to the human eye, suchas seed or plant size, or can be measured by biochemical techniques,such as detecting the protein, starch, or oil content of seed or leaves,or by observation of a metabolic or physiological process, e.g. bymeasuring tolerance to water deprivation or particular salt or sugarconcentrations, or by the observation of the expression level of a geneor genes, e.g., by employing Northern analysis, RT-PCR, microarray geneexpression assays, or reporter gene expression systems, or byagricultural observations such as hyperosmotic stress tolerance oryield. Any technique can be used to measure the amount of, comparativelevel of, or difference in any selected chemical compound ormacromolecule in the transformed or transgenic plants, however.

“Trait modification” refers to a detectable difference in acharacteristic in a plant with reduced or eliminated expression, orectopic expression, of a polynucleotide or polypeptide of the presentdisclosure relative to a plant not doing so, such as a wild-type plant.In some cases, the trait modification can be evaluated quantitatively.For example, the trait modification can entail at least a 2% increase ordecrease, or an even greater difference, in an observed trait ascompared with a control or wild-type plant. It is known that there canbe a natural variation in the modified trait. Therefore, the traitmodification observed entails a change of the normal distribution andmagnitude of the trait in the plants as compared to control or wild-typeplants.

When two or more plants have “similar morphologies”, “substantiallysimilar morphologies”, “a morphology that is substantially similar”, orare “morphologically similar”, the plants have comparable forms orappearances, including analogous features such as overall dimensions,height, width, mass, root mass, shape, glossiness, color, stem diameter,leaf size, leaf dimension, leaf density, internode distance, branching,root branching, number and form of inflorescences, and other macroscopiccharacteristics, and the individual plants are not readilydistinguishable based on morphological characteristics alone.

“Modulates” refers to a change in activity (biological, chemical, orimmunological) or lifespan resulting from specific binding between amolecule and either a nucleic acid molecule or a protein.

The term “transcript profile” refers to the expression levels of a setof genes in a cell in a particular state, particularly by comparisonwith the expression levels of that same set of genes in a cell of thesame type in a reference state. For example, the transcript profile of aparticular polypeptide in a suspension cell is the expression levels ofa set of genes in a cell knocking out or overexpressing that polypeptidecompared with the expression levels of that same set of genes in asuspension cell that has normal levels of that polypeptide. Thetranscript profile can be presented as a list of those genes whoseexpression level is significantly different between the two treatments,and the difference ratios. Differences and similarities betweenexpression levels may also be evaluated and calculated using statisticaland clustering methods.

With regard to gene knockouts as used herein, the term “knockout” refersto a plant or plant cell having a disruption in at least one gene in theplant or plant cell, where the disruption results in a reducedexpression (knockdown) or altered activity of the polypeptide encoded bythat gene compared to a control cell. The knockout can be the result of,for example, genomic disruptions, including chemically induced genemutations, fast neutron induced gene deletions, X-rays inducedmutations, transposons, TILLING (McCallum et al., 2000), homologousrecombination or DNA-repair processes, antisense constructs, senseconstructs, RNA silencing constructs, RNA interference (RNAi), smallinterfering RNA (siRNA) or microRNA, VIGS (virus induced gene silencing)or breeding approaches to introduce naturally occurring mutant variantsof a given locus. A T-DNA insertion within a gene is an example of agenotypic alteration that may abolish expression of that gene.

“Ectopic expression or altered expression” in reference to apolynucleotide indicates that the pattern of expression in, e.g., atransformed or transgenic plant or plant tissue, is different from theexpression pattern in a wild-type plant or a reference plant of the samespecies. The pattern of expression may also be compared with a referenceexpression pattern in a wild-type plant of the same species. Forexample, the polynucleotide or polypeptide is expressed in a cell ortissue type other than a cell or tissue type in which the sequence isexpressed in the wild-type plant, or by expression at a time other thanat the time the sequence is expressed in the wild-type plant, or by aresponse to different inducible agents, such as hormones orenvironmental signals, or at different expression levels (either higheror lower) compared with those found in a wild-type plant. The term alsorefers to altered expression patterns that are produced by lowering thelevels of expression to below the detection level or completelyabolishing expression. The resulting expression pattern can be transientor stable, constitutive or inducible. In reference to a polypeptide, theterms “ectopic expression” or “altered expression” further may relate toaltered activity levels resulting from the interactions of thepolypeptides with exogenous or endogenous modulators or frominteractions with factors or as a result of the chemical modification ofthe polypeptides.

The term “overexpression” as used herein refers to a greater expressionlevel of a gene in a plant, plant cell or plant tissue, compared toexpression of that gene in a wild-type plant, cell or tissue, at anydevelopmental or temporal stage. Overexpression can occur when, forexample, the genes encoding one or more polypeptides are under thecontrol of a strong promoter (e.g., the cauliflower mosaic virus 35Stranscription initiation region). Overexpression may also be achieved byplacing a gene of interest under the control of an inducible or tissuespecific promoter, or may be achieved through integration of transposonsor engineered T-DNA molecules into regulatory regions of a target gene.Thus, overexpression may occur throughout a plant, in specific tissuesof the plant, or in the presence or absence of particular environmentalsignals, depending on the promoter or overexpression approach used.

Overexpression may take place in plant cells normally lacking expressionof polypeptides functionally equivalent or identical to the presentpolypeptides. Overexpression may also occur in plant cells whereendogenous expression of the present polypeptides or functionallyequivalent molecules normally occurs, but such normal expression is at alower level. Overexpression thus results in a greater than normalproduction, or “overproduction” of the polypeptide in the plant, cell ortissue.

The term “transcription regulating region” refers to a DNA regulatorysequence that regulates expression of one or more genes in a plant whena transcription factor having one or more specific binding domains bindsto the DNA regulatory sequence. Transcription factors typically possessa conserved DNA binding domain. The transcription factors also comprisean amino acid subsequence that forms a transcription activation domainthat regulates expression of one or more abiotic stress tolerance genesin a plant when the transcription factor binds to the regulating region.

“Yield” or “plant yield” refers to increased plant growth, increasedcrop growth, increased biomass, and/or increased plant productproduction (including grain), and is dependent to some extent ontemperature, plant size, organ size, planting density, light, water andnutrient availability, and how the plant copes with various stresses,such as through temperature acclimation and water or nutrient useefficiency.

“Planting density” refers to the number of plants that can be grown peracre. For crop species, planting or population density varies from acrop to a crop, from one growing region to another, and from year toyear. Using corn as an example, the average prevailing density in 2000was in the range of 20,000-25,000 plants per acre in Missouri, USA. Adesirable higher population density (which is a well-known contributingfactor to yield) would be at least 22,000 plants per acre, and a moredesirable higher population density would be at least 28,000 plants peracre, more preferably at least 34,000 plants per acre, and mostpreferably at least 40,000 plants per acre. The average prevailingdensities per acre of a few other examples of crop plants in the USA inthe year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000;soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000and cotton 28,000-55,000 plants per acre (Cheikh et al. (2003) U.S.Patent Application No. US20030101479). A desirable higher populationdensity for each of these examples, as well as other valuable species ofplants, would be at least 10% higher than the average prevailing densityor yield.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Polypeptides and Polynucleotidesof the Disclosure

The present disclosure includes EAR mutation variants of class II HD-ZIPpolypeptides related to AtHB17, and isolated or recombinantpolynucleotides encoding these EAR mutation variants. Exemplary class IIHD-ZIP polypeptides that can be used to generate EAR mutations areprovided in the Sequence Listing; the recombinant EAR mutantpolynucleotides of the instant disclosure may be incorporated inexpression vectors for the purpose of producing transformed plants. Alsoprovided are methods for modifying plant traits including nitrogen useefficiency, photosynthetic rates, stomatal conductance, and yield. Thesemethods are based on the ability to alter the expression of HD-ZIPregulatory polypeptides that may be conserved between diverse plantspecies to promote certain phenotypes and alter the repression activityof the native HD-ZIP polypeptides by mutagenizing the EAR motif toreduce other phenotypes.

Identification of the Class II HD-ZIP Regulatory Polypeptides

Related conserved class II HD-ZIP regulatory molecules may be originallydiscovered in a model system such as Arabidopsis and homologous,functional molecules then discovered in other plant species.

Exemplary polynucleotides encoding the polypeptides of the instantdisclosure were identified in the Arabidopsis thaliana GenBank databaseusing publicly available sequence analysis programs and parameters.Sequences initially identified were then further characterized toidentify sequences comprising specified sequence strings correspondingto sequence motifs present in families of known polypeptides. Furtherexemplary polynucleotides encoding the polypeptides of the instantdisclosure were identified in the plant GenBank database using publiclyavailable sequence analysis programs and parameters.

Additional polynucleotides of the instant disclosure were identified byscreening Arabidopsis thaliana and/or other plant cDNA libraries withprobes corresponding to known polypeptides under low stringencyhybridization conditions. Additional sequences, including full lengthcoding sequences, were subsequently recovered by the rapid amplificationof cDNA ends (RACE) procedure using a commercially available kitaccording to the manufacturer's instructions. Where necessary, multiplerounds of RACE are performed to isolate 5′ and 3′ ends. The full-lengthcDNA was then recovered by a routine end-to-end polymerase chainreaction (PCR) using primers specific to the isolated 5′ and 3′ ends.Exemplary sequences are provided in the Sequence Listing.

Many of the sequences in the Sequence Listing, derived from diverseplant species, have been ectopically expressed in overexpressor plantsand have shown to confer similar traits to plants overexpressing AtHB17.

Structural Information for AtHB17 (G1543) and Related Class II HD-ZIPFamily Polypeptides

AtHB17 is a member of the class II sub-group of HD-Zip polypeptides,which is the largest group of plant homeodomain factors. Homeoboxproteins are transcription factors characterized by the presence of ahomeodomain, which usually is encoded by a conserved DNA stretch of 183base pairs specifying a 61 amino acid helix-loop-helix-turn-helix regioncapable of binding DNA as monomers or as homo- and/or heterodimers in asequence-specific manner (Affolter et al., 1990; Hayashi et al., 1990).The most conserved of three alpha helices, helix 3, binds directly intothe major groove of the DNA (Hanes and Brent, 1989). Homeodomains areknown to involve in the transcriptional regulation of key eukaryoticdevelopmental processes(www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?ascbin=8&maxaln=10&seltype=2&uid=197696).

HD-Zip polypeptides have been implicated in mediating the response ofplants to a range of environmental conditions as well as regulatingseveral developmental processes (Ariel et al., 2007). HD-Zippolypeptides have only been reported from plants (Aso et al., 1990).They contain a homeodomain linked to a leucine zipper motif, which isthought to facilitate dimerization with each other or other leucinezipper proteins and is important for the function of the polypeptides(Ruberti et al., 1991). In general, these polypeptides have beenimplicated in regulating developmental processes associated with theresponse of plants to environmental conditions (Carabelli et al., 1993;Schena et al., 1993). HD-Zip polypeptides bind as dimers topseudopalindromic recognition sites, and this interaction is dependenton the precise spacing between the DNA binding HD domain and the dimerforming leucine zipper region (Sessa et al., 1993). Random in vitrobinding site selection indicated that for HD-Zip class I proteins, thefavored recognition site was composed of two 5 base pair half sites thatoverlap at a central position (CAAT(A/T)ATTG) (Sessa et al., 1993).HD-Zip class II proteins interact with a similar nine base pair sequencebut show a preference for G/C at the central position. The central basepair preference seems to be determined by the presence of glutamine andthreonine at positions 46 and 56, respectively, in the HD of class IIproteins whereas class I proteins contain an alanine and a tryptophanresidue (Sessa et al., 1993).

Sequences in the AtHB17 subclade within the Class II HD-Zip subfamilyall comprise a putative repression domain, a homeodomain, a homeodomainassociated leucine zipper (HALZ domain), and a carboxy terminal “CPSCE”domain. At the N-terminal end of the repression domain is an EAR motifL×L×I/L/M (SEQ ID NO: 50) (FIG. 1B). The EAR motif is important forconferring transcriptional repression function to domain I of theAUX/IAA factors in the regulation of auxin response (Tiwari et al.,2004). The EAR motif shared among AtHB17 subclade sequences is closelyrelated to the EAR motif (ERF-associated amphiphilic repression motif)found in the ATHB2 subclade within the class II HD-ZIP subfamily (Ikedaand Ohme-Takagi, 2009; Ciarbelli et al., 2008; Kagale et al. 2010).ATHB2 and HAT2 have been previously shown to work as transcriptionalrepressors in vitro and in vivo (Ohgishi et al., 2001; Sawa et al.,2002; Steindler et al., 1999). AtHB17 additionally contains a uniqueN-terminal extension consisting of amino acids residues 1-73 (FIG. 1A).This extension is rich in cysteines of unknown function, which couldsuggest either DNA binding or potential sites for redox regulation. The“CPSCE” domain, which is downstream of the leucine zipper domain, refersto the five conserved amino acids Cys, Pro, Ser, Cys, Glu. In AtHB17subclade sequences as shown in FIG. 1F, the corresponding domaincomprised within AtHB17 is a “CPRCE” domain which refers to, in order,conserved Cys, Pro, Arg, Cys, and Glu amino acids. The “CPSCE” domain isreported as being responsible for redox cell state sensing (Ariel etal., 2007. Trends Plant Sci. 12: 1360-1385), and the “CPRCE” domain mayalso possess this function.

Table 1 shows a number of AtHB17 subclade sequences and includes the SEQID NO: (Column 1); the species from which the sequence was derived(Column 2) and the Gene Identifier (“GID”; Column 3), the percentidentity of the polypeptide in Column 1 to the full length AtHB17polypeptide, SEQ ID NO: 2 (Column 8), as determined by EuropeanMolecular Biology Open Software Suite (EMBOSS) Needle program (Rice, P.,et al. 2000) with a gap open penalty of 10.0 and a gap extension penaltyof 0.5; the amino acid residue coordinates for the conserved HB domains(Homeodomain), in amino acid coordinates beginning at the n-terminus, ofeach of the sequences (Column 4); the SEQ ID NO of each of the HBdomains and HALZ domains (Column 5); the conserved HB domain sequencesand the conserved HALZ domain sequences of the respective polypeptides(Column 6); and the percentage identity of the conserved domains inColumn 6 to the conserved domains of the AtHB17 sequence, SEQ ID NO: 2(Column 7).

TABLE 1 Conserved domains of AtHB17 subclade sequences Col. 7 Col. 8Percent ID Percent Col. 5 of the ID of the Conserved conserved fullCol. 4 HB domains length Col. 2 Conserved domain to sequence Col.Species HB domain and Col. 6 AtHB17 to 1 from which and HALZ HALZConserved homeo AtHB17 SEQ SEQ ID Col. 3 domain domain homeodomain andfull ID NO: was Gene ID amino acid SEQ ID and HALZ HALZ length NO:derived (GID) coordinates NO domain domains sequence  2 ArabidopsisAtHB17 136-195 51 Homeodomain: 100 100 thaliana (G1543) PPRKKLRLTREQSRLLEDS FRQNHTLNP KQKEVLAKH LMLRPRQIE VWFQNRRA RSKLKQ 196-237 70HALZ domain: 100 TEMECEYLK RWFGSLTEE NHRLHREVE ELRAIKVGPT TVNSA  3Glycine max G3524  62-121 52 Homeodomain: 88 46.3 PPRKKLRLT KEQSRLLEESFRQNHTLNP KQKESLAMQ LKLRPRQVE VWFQNRRA RSKLKQ 122-162 71 HALZ: 85.7%TEMECEYLK RWFGSLTEQ NRRLQREVE ELRAIKVGPP TVIS  4 Oryza sativa G3510 75-134 53 Homeodomain: 75 40.7 HRPKKLRLS 78.6% KEQSRLLEES FRLNHTLTPKQKEALAIK LKLRPRQVE VWFQNRRA RTKLKQ 135-175 72 HALZ domain: 81 TEMECEYLKRCFGSLTEEN RRLQREVEE LRAMRVAPP TVLS  5 Glycine max G4371  62-121 54Homeodomain: 86.7% 45.9 PPRKKLRLT KEQSLLLEES FRQNHTLNP KQKESLAMQLKLRPRQVE VWFQNRRA RSKLKQ 122-162 73 HALZ domain: 85.7% TEMECEYLKRWFGSLTEQ NRRLQREVE ELRAIKVGPP TVIS  6 Zea mays G4369  77-136 55Homeodomain: 76.7% 41.4 HRAKKLRLS KEQSRLLEES FRLNHTLTP KQKEALAVKLKLRPRQVE VWFQNRRA RTKLKQ 137-177 74 HALZ domain: 76.2% TELECEYLKRCFGSLTEEN RRLQREVEE LRAMRVAPP TVLS  7 Zea mays G4370  76-135 56Homeodomain: 74.2% 39.8% HRPKKLRLS KEQSRLLEES FRLNHTLSPK QKEALAIKLKLRPRQVEV WFQNRRART KLKH 136-176 75 HALZ domain: 76.2% TEMECEYLKRCFGSLTEEN RRLQREVEE LRAMRMAPP TVLS  8 Arabidopsis G2712  66-125 57Homeodomain: 73.3 40.1 thaliana RRRKKLRLT KEQSHLLEES FIQNHTLTPKQKKDLATFL KLSQRQVEV WFQNRRARS KLKH 126-163 76 HALZ domain: 66.7TEMECEYLK RWFGSLKEQ NRRLQIEVEE LRALKPSSTS  9 Arabidopsis G400 126-185 58Homeodomain 66.7% 31.1 thaliana (AtHB2) NSRKKLRLS KDQSAILEET FKDHSTLNPKQKQALAK QLGLRARQV EVWFQNRRA RTKLKQ 186-226 77 HALZ domain: 50.0%TEVDCEFLR RCCENLTEE NRRLQKEVT ELRALKLSP QFYMH 10 Arabidopsis G399127-186 59 Homeodomain 65.0% 29.2 thaliana TSRKKLRLS KDQSAFLEE TFKEHNTLNPKQKLALAK KLNLTARQV EVWFQNRRA RTKLKQ 187-227 78 HALZ domain: 50.0%TEVDCEYLK RCVEKLTEE NRRLQKEAM ELRTLKLSPQ FYGQ 11 Arabidopsis G398132 -191 60 Homeodomain 35.3 thaliana TCRKKLRLS KDQSAVLED TFKEHNTLNPKQKLALAK KLGLTARQV EVWFQNRRA RTKLKQ 192-232 79 HALZ domain 64.3%TEVDCEYLK RCVEKLTEE NRRLEKEAA ELRALKLSPR LYGQ 12 Sorghum SbHB17  78-13761 Homeodomain 39.7% bicolor HRSKKLRLS KEQSRLLEES FRFNHTPTPK QKEALAGKLQLRPR QVEVWFQN RRARTKLKQ 138-178 80 HALZ domain 76.2% TELECEYLKRCFGSLTEEN RRLQREVEE LRAMRVAPP TVLS 13 Vitis VvHB17  74-133 62Homeodomain 81.0% 41.9% vinifera PPRKKLRLS KDQSRLLEE SFRQNHTLN PKQKEALAMQLKLRPRQV EV WFQNRRARS KLKQ 134-174 81 HALZ domain TEMECEYLK RWFGSLTEQNRRLQREVE ELRAMKVAP PTVIS 14 Carica CpHB17  62-121 63 Homeodomain 88.346.6 papaya PPRKKLRLT KEQSRLLEES FRQNHTLNP KQKETLATQ LKLRPRQVE VWFQNRRARSKLKQ 122 -162 82 HALZ domain: 83.3% TEMECEYLK RWFGSLTEQ NRRLQREVEELRAMKVGP PTVIS 15 Populus PtHB17  56-115 64 Homeodomain 81.7% 40.5%trichocarpa PPRKK LRLSKEQSRL LEESFRQHHS LNPRQKEAL ALQLKLRPR QVEVWFQNRRARSKLKQ 116-156 83 HALZ domain: 83.3% TEMEC EYLKRWFGS LTEQNRRLQREVEELRAL KVGPPTVIS 16 Thellungiella ThHB17  60-119 65 Homeodomain:98.3% halophila PPRKKLRLT REQSRLLEDS FRQNHTLNP KQKEALAKH LMLRPRQIEVWFQNRRA RSKLKQ 120-160 84 HALZ domain: 85.7% TEMECEYLK RWFGSLTEQNHRLHREVE ELRTMKVGP PTVTS 17 Ricinus RcHB17  67-126 66 Homeodomain:83.3% 43.6% communis PPRKKLRLS KEQSRLLEES FRQHHTLNP RQKEALAM QLKLRPRQVEVWFQNRRA RSKLKQ 127-167 85 HALZ domain: 83.3% TEMECEYLK RWFGSLTEQNRRLQREVE ELRAMKVGP PTVLS 18 Arabidopsis AlHB18  68-127 67 Homeodomain:73.3 39.5 lyrata RRRKKLRLT KEQSHLLEES FIQNHTLTPK QKKDLATFL KLSQRQVEVWFQNRRARS KLKH 128-163 86 HALZ domain: 66.7% TEMECEYLK RWFGSLKEQNRRLQIEVEE LRALKPSS 19 Arabidopsis AlHB17  62-121 68 Homeodomain: 98.370.3 lyrata PPRKKLRLT REQSRLLEDS FRQNHTLNP KQKEALAKH LMLRPRQIE VWFQNRRARSKLKQ 122-162 87 HALZ domain: 92.9 TEMECEYLK RWFGSLTEQ NHRLHREVEELRAMKVGP TTVNS

Functional Characteristics of AtHB17 Subclade Sequences

35S::AtHB17 Arabidopsis plants exhibited a range of phenotypes such asdarker green leaves, altered leaf morphology, and increasedphotosynthetic capacity as compared with wild-type plants. The darkcoloration indicated that the plants likely had higher chlorophyllcontent and photosynthetic capacity. However, these plants were oftensmaller than controls.

Arabidopsis plants transformed with a G2712 polynucleotide encoding SEQID NO: 8, a closely-related paralog of AtHB17, also exhibited darkergreen leaves and a significant reduction in plant size relative tocontrol Arabidopsis plants not transformed with the G2712polynucleotide.

Sequence Variations

It will readily be appreciated by those of skill in the art that theinstant disclosure includes any of a variety of polynucleotide sequencesprovided in the Sequence Listing or capable of encoding polypeptidesthat function similarly to those provided in the Sequence Listing orTables 1. Due to the degeneracy of the genetic code, many differentpolynucleotides can encode identical and/or substantially similarpolypeptides in addition to those sequences illustrated in the SequenceListing. Nucleic acids having a sequence that differs from the sequencesshown in the Sequence Listing, or complementary sequences, that encodefunctionally equivalent peptides (that is, peptides having some degreeof equivalent or similar biological activity) but differ in sequencefrom the sequence shown in the sequence listing due to degeneracy in thegenetic code, are also within the scope of the instant disclosure.

Altered polynucleotide sequences encoding polypeptides include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polynucleotide encoding a polypeptide withat least one functional characteristic of the instant polypeptides.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding the instant polypeptides, and improper orunexpected hybridization to allelic variants, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingthe instant polypeptides.

Sequence alterations that do not change the amino acid sequence encodedby the polynucleotide are termed “silent” variations. With the exceptionof the codons ATG and TGG, encoding methionine and tryptophan,respectively, any of the possible codons for the same amino acid can besubstituted by a variety of techniques, for example, site-directedmutagenesis, available in the art. Accordingly, any and all suchvariations of a sequence selected from the above table are a feature ofthe instant disclosure.

In addition to silent variations, other conservative variations thatalter one, or a few amino acids in the encoded polypeptide, can be madewithout altering the function of the polypeptide. For example,substitutions, deletions and insertions introduced into the sequencesprovided in the Sequence Listing are also envisioned. Such sequencemodifications can be engineered into a sequence by site-directedmutagenesis (for example, Smith et al, 1992) or the other methods knownin the art or noted herein Amino acid substitutions are typically ofsingle residues; insertions usually will be on the order of about from 1to 10 amino acid residues; and deletions will range about from 1 to 30residues. In preferred embodiments, deletions or insertions are made inadjacent pairs, for example, a deletion of two residues or insertion oftwo residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a sequence. The mutations that aremade in the polynucleotide encoding the transcription factor should notplace the sequence out of reading frame and should not createcomplementary regions that could produce secondary mRNA structure.Preferably, the polypeptide encoded by the DNA performs the desiredfunction.

Conservative substitutions are those in which at least one residue inthe amino acid sequence has been removed and a different residueinserted in its place. Such substitutions generally are made inaccordance with the Table 2 when it is desired to maintain the activityof the protein. Table 2 shows amino acids which can be substituted foran amino acid in a protein and which are typically regarded asconservative substitutions.

TABLE 2 Possible conservative amino acid substitutions Amino AcidResidue Conservative substitutions Ala Ser Arg Lys Asn Gln; His Asp GluGln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; ValTrp Tyr Tyr Trp; Phe Val Ile; Leu

Similar substitutions are those in which at least one residue in theamino acid sequence has been removed and a different residue inserted inits place. Such substitutions generally are made in accordance with theTable 3 when it is desired to maintain the activity of the protein.Table 3 shows amino acids which can be substituted for an amino acid ina protein and which are typically regarded as structural and functionalsubstitutions. For example, a residue in column 1 of Table 3 may besubstituted with residue in column 2; in addition, a residue in column 2of Table 3 may be substituted with the residue of column 1.

TABLE 3 Similar amino acid substitutions Residue Similar substitutionsAla Ser; Thr; Gly; Val; Leu; Ile Arg Lys; His; Gly Asn Gln; His; Gly;Ser; Thr Asp Glu, Ser; Thr Gln Asn; Ala Cys Ser; Gly Glu Asp Gly Pro;Arg His Asn; Gln; Tyr; Phe; Lys; Arg Ile Ala; Leu; Val; Gly; Met LeuAla; Ile; Val; Gly; Met Lys Arg; His; Gln; Gly; Pro Met Leu; Ile; PhePhe Met; Leu; Tyr; Trp; His; Val; Ala Ser Thr; Gly; Asp; Ala; Val; Ile;His Thr Ser; Val; Ala; Gly Trp Tyr; Phe; His Tyr Trp; Phe; His

The Sequence Listing provides polypeptides that have regulatoryactivities. Although all conservative amino acid substitutions (forexample, one basic amino acid substituted for another basic amino acid)in a polypeptide will not necessarily result in the polypeptideretaining its activity, it is expected that many of these conservativemutations would result in the polypeptide retaining its activity. Mostmutations, conservative or non-conservative, made to a protein butoutside of a conserved domain required for function and protein activitywill not affect the activity of the protein to any great extent.

The Effects of the EAR Mutation on AtHB17 Function

The class II HD-ZIP polypeptides described herein all contain EARmotifs, within which are two highly conserved leucine residues (FIG.1B). Our experiments showed that the conserved leucine residues withinthe EAR motif are required for AtHB17's transcriptional repressionactivity in protoplasts.

To analyze the physiological relevance of the EAR mutation, nucleic acidconstructs encoding AtHB17, SEQ ID NO: 2, or AtHB17 EAR mutants (i.e.,AtHB17 L84A L86A, a mutant contains the L84A and L86A mutations in theEAR motif, SEQ ID NO: 1), or the chloramphenicol acetyltransferase (CAT,SEQ ID NO: 24) were co-transfected with the prHAT1::GUS reporter geneconstruct into Arabidopsis mesophyll cell protoplasts to analyze therepression activity. The results show in comparison to the control group(CAT), AtHB17 was able to repress GUS reporter expression, while AtHB17EAR mutant lost that repression activity (FIG. 8). This demonstratesthat mutating the conserved leucine residues within the EAR motif ofAtHB17 caused a loss of repression. The effect of EAR mutations on theDNA-binding of AtHB17 was also examined. We were unable obtainsufficient quantity of the full length protein AtHB17 from E. coli toperform the binding assay. However, a deletion mutant of AtHB17 whichlacks amino acids 1-73 (AtHB Δ1-73) was found to express well in E. coliand also retain a significant portion of the repression activity of theAtHB17 full length polypeptide. Thus, the truncated versions of theAtHB17 and AtHB17 EAR mutation variants were used in the DNA-bindingassay. FIG. 9 shows a deletion variant of AtHB17 containing the EARdomain mutations (AtHB17 Δ1-73 L84A L86A, a mutant contains the L84A andL86A mutations and lacking the N terminal amino acid residues 1-73) wasable to bind to a DNA fragment containing a class II HD-ZIP specificbinding site (SEQ ID NO:48) to an extent comparable to a deletion mutantAtHB17 Δ1-73 (a mutant which lacks amino acid residues 1-73 compared tothe full length polypeptide, AtHB17, SEQ ID NO: 2). These results showthat mutations in the EAR domain (e.g., L84A, L86A) dramatically reducerepression activity without impairing the DNA binding of AtHB17. It isvery likely that the EAR mutations relieve repression of the targetgenes through blocking the recruitment of other co-repressors such asGroucho/Tup1 proteins (Hill K. et al., 2008 Plant J 53, 172-185;Szemenyei H. et al., 2008. Science 319, 1384-1386).

When overexpressed in Arabidopsis, AtHB17(L84A L86A) produced similarphenotypes to those produced by overexpressing AtHB17, such as increasedphotosynthetic capacity, higher chlorophyll content, altered leafmorphology, increased photosynthetic rates, reduced petiole length, andearly flowering compared with wild-type plants. This indicates that therepression activity of the AtHB17 is not required for the enhancedphotosynthetic capacity caused by AtHB17. Significantly, unlike the fulllength AtHB17, overexpression of AtHB17 (L84A L86A) did not result inthe dwarf phenotypes (FIG. 7B) in Arabidopsis.

It is expected that mutations substituting the conserved leucines in theEAR motif of AtHB17 with any amino acid other than isoleucine and valine(which are conservative substitutes for leucine) in AtHB17 will havesimilar effect as the leucine to alanine substitution present in AtHB17(L84A L86A). It is also expected that mutagenizing the leucines in theEAR motifs of other AtHB17 clade members will have similar effect on theactivities of these clade members.

This specification describes methods to introduce mutations into the EARmotifs of the polynucleotides encoding class II HD-ZIP polypeptides thatare similar to AtHB17 in structure and function. The EAR mutationvariants of these class II HD-ZIP polypeptides then can be used toconfer certain traits, e.g., greater nitrogen use efficiency, greaterbiomass, greater size, greater photosynthetic rates and greater yield inC3 or C4 plants, while reducing other phenotypes (for example, smallerplant size). This method can be used in a wide range of speciesincluding major row crops (e.g. soy, corn, cotton, canola, sugarcane,wheat, alfalfa, sugarbeet, and vegetables), forestry, and bioenergygrasses (e.g. Miscanthus or switchgrass). EAR mutant forms of class IIHD-ZIP could also be deployed to produce increased photosyntheticcapacity to species such as algae.

EXAMPLES

It is to be understood that this instant disclosure is not limited tothe particular devices, machines, materials and methods described.Although particular embodiments are described, equivalent embodimentsmay be used to practice the instant disclosure.

The instant disclosure, now being generally described, will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present disclosure and are not intended to limit thespecification or claims. It will be recognized by one of skill in theart that a polypeptide that is associated with a particular first traitmay also be associated with at least one other, unrelated and inherentsecond trait which was not predicted by the first trait.

Example I. Producing and Cloning the EAR Mutation in AtHB17

A full-length AtHB17 clone was subject to primer-based site-directedmutagenesis, applying oligonucleotides of the sequences,5′-CGACGGCGGCCGCAATGGCGATTTTGCCGGAAAA-3′ (SEQ ID NO: 90) and5′-GTGCGCCCGGGTCAACGATCACGCTCTTGCGGC-3′ (SEQ ID NO: 91), according tothe directions of the QuikChange site-directed mutagenesis kit(Invitrogen) to introduce a double point mutation L84A and L86A in thefull length sequence. The EAR mutations were confirmed by sequencingreactions and the resulting mutant polynucleotide AtHB17 L84A L86A (SEQID NO: 46) was cloned into pMEN65 with native 5′ and 3′ AtHB17 UTRs bySalI/NotI restriction enzymes and transformed into the wild-typeColumbia ecotype of Arabidopsis thaliana.

Example II. Transformation of the EAR Mutant Polynucleotide Sequences

Arabidopsis plants were transformed by the floral dip method (Clough andBent, 1998) using Agrobacterium carrying a transformation constructwhich contained a kanamycin resistance selectable marker system drivenby the NOS promoter and the AtHB17 clone downstream from the CaMV 35Spromoter. In all experiments, a control line generated by transformingCol-0 with the empty transformation construct lacking the AtHB17 clonewas used to determine the effect of AtHB17 overexpression. Betweentwenty and forty independent primary transformants were isolated onselection media. Transformants were PCR-genotyped to confirm that theyharbored the correct transgene. Expression of AtHB17 mRNA transcriptswas verified by RT-PCR on RNA extracted from leaves of 40 day oldplants. Lines that showed substantial levels of overexpression versusthe control line were selected and used in subsequent experiments.

Example III. Protoplast-Based Transcriptional Repression Assays

Protoplast Transfection

Arabidopsis protoplasts were isolated and transfected as previouslydescribed (Tiwari et al. 2006). Protoplasts were prepared from wholeleaves of 3-4 weeks old Arabidopsis plants using an enzyme solutioncontaining cellulose (Research Products International Corp) andmacerozyme (SERA Electrophoresis GmbH) to remove cell walls. Plasmid DNAfor transfection was prepared using Qiagen EndoFree Plasmid maxi kits.DNA was introduced by incubating protoplasts with 40% PEG for 30minutes. Following removal of PEG, protoplasts were kept in the dark atroom temperature for 18-20 hours.

Reporter Activity Assay

To assess the repression activity of the variants, 200 μl of protoplastcells were transfected with 10 μg of plasmid DNA of the GUS reporterconstruct with 5 μg of plasmid DNA of effector protein constructs,including the EAR mutants and native AtHB17. A 35S::CAT construct wasused as a negative control. Following overnight incubation, cells werelysed in 1× cell culture lysis buffer (Promega) and incubated with 1 mMMUG (GBT) solution at 37° C. for 1 hour. GUS activity was quantified bytaking fluorescence measurements using a Synergy HT Microplate Reader(BioTek). Three biological replicated were performed and GUSmeasurements for each construct were averaged.

In Vitro DNA-Binding Assay

To determine the effects of EAR mutation on the DNA-binding activity ofAtHB17, DNA binding assays were performed in a 96-well plate-basedformat using the NoShift Transcription Factor Assay kit according to themanufacturer's protocol (Novagen). Duplex DNA was prepared by annealingsense and antisense 3′-biotin-TEG oligonucleotides in 0.5×SSC at a finalconcentration of 10 pmol/μL. Protein-DNA interactions were assayed invitro using 10 ng recombinant protein (from soluble lysate) in 1×NoShift Binding Buffer, 0.01 U poly(dI-dC)-poly(dI-dC), 25 μg salmonsperm DNA and 10 pmol of duplex DNA in a final volume of 20 μL. Theprotein-DNA complex was allowed to form by incubation on ice for 30 min.The protein-DNA complex was then diluted five-fold with 1× NoShiftbinding buffer and added to a streptavidin-coated plate, which wasincubated for one hour at 37° C. After the binding incubation, the platewas incubated with primary antibody, either α-Penta-His HRP-conjugatedantibody (1:1000, Qiagen) or the AtHB17 polyclonal antibody (1:1000,Monsanto) for one hour at 37° C. For the AtHB17 polyclonal antibody,incubation with HRP-conjugated secondary goat anti-rabbit antibody(1:10000, Thermo Scientific) for one hour at 37° C. was required. Aseries of washes with gentle agitation followed each incubation step. Todevelop colorimetric signal, 1-Step Ultra TMB-ELISA(3,3′,5,5′-tetramethylbenzidine) (Thermo Scientific) was added to theplate wells and incubated for 15 min. Hydrochloric acid was added tostop the reaction and to provide greater sensitivity. Binding was thenquantified by measurement of the absorbance at 450 nm using a Synergy HTMicroplate Reader (Bio-Tek).

The DNA-binding elements used for this set of AtHB17 activity assays aresummarized below (consensus binding sites are underlined):

(SEQ ID NO: 48) CAGACAATCATTGCGGC = HD-ZIP Class II consensus (1 repeat)SEQ ID NO: 49) CAGATCAGTCTGACGGC = Mutated HD-ZIP Class IIconsensus (1 repeat)

Example III. Arabidopsis Plant Growth Conditions andPhotosynthesis-Related Physiological Assays

Seeds of AtHB17 lines, AtHB17 EAR mutant lines, and empty vectorcontrols (pMEN65) were chlorine gas sterilized and sown onto plates with80% MS+1% sucrose media. Plates were stratified in the dark at 4° C. for3 days, then moved to a growth chamber (ATC 26, Controlled EnvironmentsLtd, Winnipeg, Manitoba, Canada) operating a 10 hour photoperiod at aphotosynthetic photon flux (PPF) of ca. 150 μmol m−2 s−1 at plantheight, and a 22° C. day/19° C. night, air temperature (Tair). Afterseven days, seedlings were transplanted into autoclaved ProMix soil inplastic pots and returned to the same growth chamber. To minimizeproblems associated with small gradients in environmental conditionswithin the chamber, plants from different lines being compared in agiven experiment were mixed within flats, and flat positions wererotated weekly within, the growth chamber. All flats were bottom wateredthree times a week, once with water containing a Peter's fertilizersolution (20-20-20 NPK; 0.8 g/L).

Leaf Photosynthesis-Related Physiological Analysis

After 35 days growth in the conditions described above, photosyntheticcapacity, photosynthetic rate and stomatal conductance were measured.These measurements were determined from measurements of leaf CO₂ uptakeand H₂O loss made using an infra-red gas analyzer (LI-6400 XT, LicorInc, Lincoln, NB, USA). All measurements were made after 40-50 minacclimation of whole Arabidopsis rosettes to a photosynthetic-photonflux (PPF) of 700 mmol m⁻² s⁻¹, under LED light banks emitting visiblelight (SL3500 Warm White 400-700 nm, Photon Systems Instruments, Brno,Czech Republic). The protocol of Long and Bernacchi (2003) served as areference for creating plots of light-saturated photosynthesis (A_(sat))and the rate of linear electron transport through photosystem two(J_(PSII)), to sub-stomatal [CO₂] (C_(i)). Simultaneous measurements oflight-saturated leaf CO₂ uptake, H₂O loss and yields of chl afluorescence measurements were made over a range of [CO₂] from 50 to1000 μmol CO₂ mol⁻¹. Measurements were made at the growth airtemperature of 22° C. and a leaf-air water vapor pressure deficit of ca.0.8 kPa. The curve-fitting software of Sharkey et al. (2007) was used tomake in vivo estimates of: the maximum rate of RuBP-saturatedphotosynthesis (V_(c,max)), and the light-saturated capacity for RuBPregeneration, calculated and expressed in terms of electron flowrequired to support the modeled rate of A_(sat) (T_(max)). Estimates oflinear electron flow through photosystem II (J_(PSII)) were estimated asthe product of the operating efficiency of PSII (F′_(q)/F′_(m)) and thefraction of incident PAR absorbed by PSII (Genty et al., 1989). Thefraction of the 700 μmol m⁻² s⁻¹ of photosynthetically active radiation(PAR) incident upon the leaf transmitted through the leaves, wasmeasured by placing the center of the abaxial side of the leaf upon aquantum sensor (LI 190, Licor Inc.). Both the leaf and the quantumsensor were then pressed up against the upper gasket under the lightsource, so that transmission of the PAR from the light source wasmeasured. No measure of reflectance from the adaxial leaf surface wasmade; consequently, absorption was calculated as 1 minus transmissionfor the determination of J_(PSII). In common with most studies, weassumed that 50% of incident PAR was absorbed by PSII for the purposesof calculating J_(PSII). A second estimate of PAR transmission throughthe leaf was made immediately after the high-light measurement using aSPAD meter (SPAD 502, Konica Minolta Sensing Inc, Japan). Two SPADmeasurements were made per leaf, each approximately half way along theleaf, on either side of the mid rib.

Example IV. Nitrogen Use Efficiency Assays

Arabidopsis Plant Material and Growth Conditions

Seeds were surface sterilized using chlorine gas, plated on selectivemedia, and stratified for three days at 4° C. in the dark. Plates wereincubated at 22° C. under a light intensity of approximately 100 μmolem⁻² sec⁻¹ for seven days. Seedlings were transplanted into square pots(60 mm×60 mm) containing fritted clay topped with a small (10 mm) layerof medium particle sized sand and kept covered with a plastic dome foranother seven days to maintain humidity while they became established.Plants were grown on soil under fluorescent lights, at a light intensityof approximately 100 μmole m⁻² sec⁻¹ and a temperature of 22° C. (L:D10:24). Plants were cultivated under low N (2 mM nitrate) or high N (10mM nitrate) conditions. Phosphate (0.25 mM), sulphate (0.25 mM),magnesium (0.25 mM), and sodium (0.20 mM) were present in both solutionsat the same concentration. The only other differences between low andhigh N solutions were in potassium (5.25 mM and 2.75 mM in high and lowN solutions, respectively), calcium (2.50 mM and 0.50 mM, respectively),and chloride ions (0.25 mM and 0.70 mM, respectively). Pots were wateredthree times daily every eight h using a commercial ebb and flowhydroponic system (Bigfoot, American Hydroponics, Arcata Calif.).

¹⁵N Labeling and Harvest

¹⁵N uptake was estimated 32 days after sowing when plants were stillvegetative. The unlabelled watering solution was replaced by an¹⁵N-containing solution that had the same nutrient composition exceptthat ¹⁴NO₃ was replaced by ¹⁵NO₃ at 2.5% enrichment (Chardon et al.,2010; Lemaitre et al., 2008). All pots were watered for 24 h byimmersing the base of the pot with a volume of labeled solutionssufficient to cover the lower 35 mm of the pot. After 24 h, the rosettewas separated from its root to stop ¹⁵N uptake. Rosettes were then driedand their dry weight was determined. Four to six replicates wereharvested for uptake and remobilization experiments.

Determination of Total Nitrogen Content and ¹⁵N Abundance

After drying and weighing each sample, material was ground in a beadmill to obtain a homogenous fine powder. A subsample of 2 to 3 mg wascarefully weighed into tin capsules to determine the total N content and¹⁵N abundance at the Stable Isotope Facility at UC Davis. Samples wereanalyzed for ¹⁵N isotopes using a PDZ Europa ANCA-GSL elemental analyzerinterfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (SerconLtd., Cheshire, UK). Samples were combusted at 1000° C. in a reactorpacked with chromium oxide and silvered cobaltous/cobaltic oxide.Following combustion, oxides were removed in a reduction reactor(reduced copper at 650° C.). The helium carrier then flowed through awater trap (magnesium perchlorate). N₂ and CO₂ were resolved on aCarbosieve GC column (65° C., 65 mL/min) before entering the IRMS.During analysis, samples were interspersed with several replicates of atleast two different laboratory standards. These laboratory standards(selected to be compositionally similar to the samples being analyzed)were previously calibrated against NIST Standard Reference Materials(IAEA-N1, IAEA-N2, IAEA-N3, USGS-40, and USGS-41). A sample'spreliminary isotope ratio is measured relative to reference gasesanalyzed with each sample. These preliminary values are finalized bycorrecting the values for the entire batch based on the known values ofthe included laboratory standards. The ¹⁵N abundance was calculated asatom percent (A %=(¹⁵N)/(¹⁵N+¹⁴N)) and for unlabelled plant controls (A%_(control)) was 0.3660. The ¹⁵N enrichment (E %) of the plant materialwas then defined as E %=A %_(sample)−A %_(control) (Chardon et al.,2010; Lemaitre et al., 2008).

NUE was determined using the ratio of rosette biomass to N concentrationin the rosette (termed “usage index”; Good, Shrawat and Muench, 2004).By calculating NUE in this manner the absolute amount of biomassproduced as well as the ratio of biomass per unit nitrogen was factoredin. The efficiency by which N was taken up and transported to rosetteleaves relative to the rosette biomass was estimated by calculating theamount of ¹⁵N in the tissue. Multiplication by 100 converts the units tomicrograms. The amount of ¹⁵N per plant was determined by multiplyingthe NupE by tissue dry weight.

NUE (as calculated by determining the Usage Index (UI))

NUE=Usage index (UI)=DM/N %

N uptake efficiency (NupE)

NupE=¹⁵N/DM=E %×N %×100

μg ¹⁵N/plant

¹⁵N/plant=E %×N %×DM×100

DM=Dry matter

Example V. Benefits of Overexpressing an EAR Mutation Variant of AtHB17in Arabidopsis

Enhanced Photosynthetic Rate and Photosynthetic Chlorophyll UseEfficiency

The EAR mutation variant of AtHB17 demonstrated the ability to increasephotosynthetic capacity and increase photosynthetic rates whenoverexpressed in Arabidopsis plants compared to control plants. In oneof the experiments, two transgenic lines overexpressing the EAR mutationvariant (AtHB17 L84A L86A), two lines overexpressing the native AtHB17,and control plants comprising the vector pMEN65, were analyzed forchlorophyll content and photosynthesis (FIG. 2 and Table 4). Both EARmutation variant lines had higher SPAD values compared to empty vectorcontrols but lower SPAD values when compared to native AtHB17overexpressors, indicating that they possessed intermediate chlorophyllcontent levels between the empty vector control plants and the nativeAtHB17 overexpressors (Table 4). Although these EAR mutation variantoverexpressors had lower chlorophyll content compared to native AtHB17overexpressors, they did maintain the positive benefits of AtHB17overexpression by having higher light-saturated photosynthetic ratesrelative to the empty vector control plants (FIG. 2), indicating a moreoptimal investment of chlorophyll content with respect to the rate ofphotosynthesis. These findings indicate that possible dwarfing effectsresulted from constitutive AtHB17 overexpression in some species may beavoided while still retaining other phenotypes such as elevatedphotosynthesis by expression of a protein variant of AtHB17 with amutated EAR domain, or a similar variant of a homologous protein.

TABLE 4 The fraction of incident PPF transmitted through the leaf(Transmission), and estimate of chlorophyll content (SPAD), the CO₂ andlight-saturated rate of photosynthesis (A_(max)), the light-saturatedrate of photosynthesis measured at an atmospheric [CO₂] of 400 μmolmol⁻¹ (A_(sat 400)), the CO₂ and light-saturated rate of linear electronflow though PSII (J_(PSII max)) and an estimate of the maximum capacityfor linear electron flow contributing to RuBP regeneration (J_(max)) for35S::AtHB17 protein variants and controls. Transmission SPAD A_(max)A_(sat(400)) JPSII J_(max) pMEN65 0.09 30.02 18.09 11.48 104.5 95.1(0.004) (1.10) (1.04) (1.17) (2.0) (4.0) 35S::AtHB17 373- 0.033 50.7923.39 14.11 134.5 120.9 22 (0.005)* (1.05)* (0.58)* (0.33)* (3.5)*(5.9)* 35S::AtHB17 378-6 0.055 37.92 22.38 13.93 125.9 113.8 (0.00)*(1.15)* (0.70)* (0.49)* (3.9)* (4.4)* EAR Z193359-14 0.070 33.74 20.2514.80 116.1 112.2 (0.004)* (0.42)* (0.69)* (0.46)* (2.8)* (4.2)* EARZ193358-14 0.072 33.61 22.09 14.39 119.5 109.9 (0.002)* (0.99)* (0.56)*(0.58)* (2.8)* (2.4)* Note: All data are the mean ± 1 standard error ofmeasurements made on leaves of at least six replicate plants. Thepresence of an asterisk signifies that the mean for a given line issignificantly different from the pMEN65 control (p < 0.05).Increased Photosynthetic Capacity and Increased Stomatal ConductanceResulted in Increased Photosynthetic Rate

Stomata regulate the exchange of CO₂ and H₂O, between the leaf and theatmosphere. Current understanding of stomatal physiology predicts thatleaves with higher photosynthetic capacity would have higher stomatalconductance under optimal conditions. A mechanism for indirect sensingof photosynthetic capacity underlies this relationship, with the resultthat stomata respond to maintain the CO₂ concentration inside the leafwithin a narrow range. Consequently plants with increased photosyntheticcapacity would be expected to have higher stomatal conductance. Plantsoverexpressing the EAR mutation variant (AtHB17 L84A L86A) supportedthis theory (FIGS. 2 and 3). Results from at least five independentexperiments showed stomatal conductance of 35S::AtHB17 EAR mutant lineswas never lower, and typically higher, than for controls (FIGS. 3 and4). The result was that increased photosynthetic capacity was realizedas an increased photosynthetic rate at current atmospheric CO₂concentrations in the EAR variant lines (FIG. 5). In contrast, inArabidopsis lines constitutively overexpressing the native protein,stomatal conductance is typically lower than controls (FIGS. 3 and 4),and photosynthesis may be constrained to control plant levels (FIG. 5).The EAR domain appears to have a direct or indirect effect on guard cellphysiology that is distinct from other phenotypes of 35S::AtHB17expression in Arabidopsis. This effect manifests itself as an increasedphotosynthetic rate when the AtHB17 EAR mutant is constitutivelyoverexpressed in Arabidopsis.

Reduced Transcriptional Effect on Genes Involved in Stomatal Developmentand Function Relative to the Native AtHB17

Plants overexpressing the EAR mutation variant (AtHB17 L84A L86A) showedreduced effects on gene transcription when compared to plantsoverexpressing the native AtHB17 polypeptide. Overexpression of the fulllength native form AtHB17 has a broad effect on gene expression. Incontrast, the transcriptional effects observed in lines overexpressingan EAR mutation variant of AtHB17 were dramatically reduced.Consequently, relatively few pathways were significantly perturbed inlines overexpressing EAR mutation variants than in the 35S::AtHB17lines. These findings are consistent with the EAR mutation resulting ina loss of repression activity. In particular, the transcriptionaleffects on a number of genes involved in the development of stomata orregulation of stomatal function were either diminished or lost in EARvariant lines, suggestive of differential effects on stomatal functionand related processes such as photosynthesis. For example, theexpression of numerous genes known to be involved in the regulation ofstomatal opening and closure were found to be affected in different waysby overexpression of the full length wild-type AtHB17 protein comparedto the EAR protein variant (FIG. 6). The expression of BGL1, KAT1 andKAT2 were significantly repressed in 35S::AtHB17 lines; however, theirexpression was close to control levels in EAR mutant lines (FIGS. 6A and6B). BGL1 (AtBG1), encoding a beta-glucosidase, is thought to regulatestomatal movement by producing active ABA through hydrolyzingglucose-conjugated, biologically inactive ABA. Loss of AtBG1 causesdefective stomatal movement (Lee et al., 2006). KAT1 and KAT2,Arabidopsis K⁺-channel-encoding genes expressed in guard cell, have beenfound to be critical to the opening of stomata and are induced by bluelight and circadian rhythm (Lebaudy et al., 2008; Sata et al., 2010).Similarly, loss of repression in the expression of RD20, ABI1, MYB60 andCPK6 was observed in 35S::AtHB17 L84A L86A overexpressing lines. RD20,which encodes a calcium binding protein, plays an important role indrought tolerance through closing stomates during water deficit. Theloss-of-function mutation, rd20, causes plants to exhibit increasedstomatal opening and an elevated transpiration rate, as well as areduced tolerance to drought as compared with the wild-type (Aubert etal., 2010). ABI1, encoding a phosphatase 2C protein, is a negativeregulator of ABA-mediated stomatal closure (Gosti et al., 1999). Inaddition, loss of repression was observed for MYB60 and CPK6 in EARmutant lines (FIG. 6D). Arabidopsis MYB60 is specifically expressed inguard cells. A loss of function mutation in this gene results in theconstitutive reduction of stomatal opening, and decreased wilting underwater stress conditions (Cominelli et al., 2005). CPK6 has been reportedto have an important role in guard cell ion channel regulation andstomatal movement (Mori et al., 2006).

Overexpression of EAR Mutant Variant of AtHB17 Increased Plant Size andPlant Biomass.

Previous experiments showed that overexpression of AtHB17 in Arabidopsisreduced plant growth and size. Further experiments showed thatoverexpression of the EAR mutation variant AtHB17 L84A L86A had no suchadverse effects; on the contrary, these EAR mutation variantoverexpressors were often larger and had greater biomass than controlplants (FIG. 7B). SPAD analysis indicated overexpression of the AtHB17EAR mutants moderately increased chlorophyll content levels; thechlorophyll levels of these EAR mutants were between those of thecontrol plants and of the AtHB17 overexpressors (FIG. 7A).

Overexpression of EAR Mutant Variant of AtHB17 Increased Plant NitrogenUse Efficiency (“NUE”).

Experimental results in Table 5 showed that overexpression of EAR mutantvariant significantly increased plant nitrogen use efficiency inArabidopsis, more than overexpression of native AtHB17 did (Col. 7). Inthese experiments, a number of 35S::AtHB17 lines (“378-1”, “373-22” and“370-23”) and 35S::AtHB17 L84A L86A (EAR mutant variant lines of“Z193346-14”, “Z193358-14”, and “Z193359-1”) lines were analyzed fornitrogen uptake and assimilation according to Example IV. When grownunder nitrogen-limiting conditions (2 mM NO₃), 35S::AtHB17 and35S::AtHB17 L84A L86A lines had increased NUE compared to the emptyvector control line (pMEN65). 35S::AtHB17 lines accumulated more biomass(shown in Col. 3) with less nitrogen content (shown in Col. 4), and35S::AtHB17 L84A L86A lines accumulated more biomass using the sameamount of nitrogen as that found in the empty vector control lines(pMEN65). When grown under ample nitrogen conditions (10 mM NO₃), onlythe 35S::AtHB17 L84A L86A lines utilized nitrogen more efficiently thanthe empty vector control lines by accumulating the equivalent or morebiomass with less nitrogen (Col. 7, the last three rows).

TABLE 5 Col. 4 Col. 6 Col. 3 Nitrogen Nitrogen Col. 2 Tissue Dry contentCol. 5 Uptake Col. 7 Col. 1 Plant lines weight (g) (% N) ¹⁵N/plantEfficiency NUE  2 mM pMEN65 0.010 ± 0.001 6.6 ± 0.2 67 ± 8   646 ± 8570.15 ± 0.01 AtHB17 line 0.021 ± 0.004** 6.0 ± 0.2** 59 ± 34  3202 ±1144*  0.33 ± 0.06** 378-1 AtHB17 line 0.013 ± 0.002 5.6 ± 0.4** 62 ± 114438 ± 808 0.252 ± 0.03* 373-22 AtHB17 line 0.018 ± 0.002** 5.9 ± 0.5**87 ± 16  4057 ± 1198*  0.29 ± 0.05** 370-23 AtHB17 EAR 0.030 ± 0.003**6.6 ± 0.4  297 ± 103**  7875 ± 1358*  0.46 ± 0.03** line Z193346- 14AtHB17 EAR 0.030 ± 0.004** 6.6 ± 0.7  267 ± 23**  8697 ± 1476  0.47 ±0.09** line Z193358- 14 AtHB17 EAR 0.026 ± 0.002** 6.7 ± 0.6  202 ± 13**7752 ± 828   0.39 ± 0.05** line Z193359-1 10 mM pMEN65 0.017 ± 0.002 7.5± 0.2 76 ± 7  6379 ± 683 0.20 ± 0.04 AtHB17 line 0.017 ± 0.003 6.2 ±0.4** 44 ± 20  2309 ± 779** 0.27 ± 0.05 378-1 AtHB17 line 0.012 ± 0.0025.8 ± 0.2** 61 ± 15 6280 ± 751 0.20 ± 0.02 373-22 AtHB17 line 0.011 ±0.003 6.2 ± 0.3** 91 ± 29 5163 ± 346 0.15 ± 0.03 370-23 AtHB17 EAR 0.022± 0.005 6.4 ± 0.3**  162 ± 63**  8622 ± 1745  0.35 ± 0.04** lineZ193346- 14 AtHB17 EAR 0.025 ± 0.002** 6.5 ± 0.3** 70 ± 24 2754 ± 465 0.37 ± 0.07** line Z193358- 14 AtHB17 EAR 0.022 ± 0.006 6.6 ± 0.4** 111± 22  4401 ± 805  0.34 ± 0.08** line Z193359-1 Significantly differentfrom control at: *p < 0.1, **p < 0.05

The AtHB17 Δ21 polypeptide, SEQ ID NO: 92, is identical to AtHB17, SEQID NO: 2, except for a deletion of amino acids 1-21 of the lattersequence. AtHB17 Δ21 has identical structural domains to AtHB17 Δ21,namely, the repression domain, the homeodomain, the homeodomainassociated leucine zipper (HALZ domain), and the carboxy terminal“CPRCE” domain. The EAR mutant variant of AtHB17 Δ21 has also beenconstructed by introducing a leucine to alanine substitution at position63 and 65.

It is expected that the EAR mutant variant of AtHB17 Δ21, SEQ ID NO: 93,will also confer modified traits as does the EAR mutant variant ofAtHB17, SEQ ID NO: 21, when it is overexpressed in plants. These traitsinclude, but are not limited to, higher nitrogen use efficiency, greatersize, greater biomass, greater yield, greater growth rate, greaterchlorophyll levels, greater photosynthetic capacity, greaterphotosynthetic rate and greater stomatal conductance.

It is also expected that the EAR mutation variant of AtHB17 Δ21, SEQ IDNO: 93 will have reduced transcriptional effect on genes involved instomatal development and function relative to the native AtHB17 Δ21, SEQID NO: 92. Plants expressing SEQ ID NO: 93 will also have highernitrogen use efficiency, greater stomatal conductance, lower chlorophylllevels, greater size, greater biomass, greater yield, and a greatergrowth rate relative to plants overexpressing SEQ ID NO: 92.

Example VI. Expression and Analysis of Increased Photosynthesis andIncreased Yield in Non-Arabidopsis C3 or C4 Plants

The EAR mutant variant of AtHB17 has been shown to confer increasedphotosynthetic rate, increased stomatal conductance, moderatelyincreased chlorophyll content, increased plant size and/or increasedbiomass when the EAR mutant variant of AtHB17 was overexpressed inArabidopsis. Enhanced rates of photosynthesis are expected to result inincreased crop yield in field conditions, including both C3 and C4 cropplants (Zhu et al., 2010). It is expected that similar EAR mutantsderived from structurally similar orthologs of the AtHB17, such as thoseprovided in the Sequence Listing, will confer similar improved traits incrop plants while eliminating the growth penalty associated withoverexpression of the native AtHB17 homologs.

Overexpression of EAR mutation variants is expected to be especiallybeneficial for C3 plants, whose carbon fixation pathway is moresensitive to CO₂ limitation than C4 plants, The AtHB17 EAR mutant isideally suited to C3 crops being grown on acres where water stress ismodest and infrequent, where the benefits of increased photosyntheticcapacity can be fully realized as increased photosynthetic rate. EARmutation variants of the AtHB17 homologs can be employed to enhanceplant photosynthesis, plant biomass, plant size, or yield in C3 plants,which include, but are not limited to, soybean, cotton, canola, rice,wheat, poplar, eucalyptus, and alfalfa.

After a C3 or C4 plant, or a C3 or C4 plant cell has been transformed(and the latter plant host cell regenerated into a plant) and shown tohave greater size, greater photosynthetic rate at atmospheric CO2condition, greater stomatal conductance, the transformed C3 or C4 plantmay be crossed with itself or a plant from the same line, anon-transformed or wild-type C3 or C4 plant, or another transformed C3or C4 plant from a different transgenic line of plants.

Northern blot analysis, RT-PCR or microarray analysis of theregenerated, transformed plants may be used to show expression of an EARmutant class II HD-ZIP polypeptide of the instant disclosure and relatedgenes that are capable of conferring greater photosynthetic rate, higherstomatal conductance and/or larger size.

The ectopic overexpression of EAR mutation variants of these AtHB17homologous sequences may be regulated using constitutive, inducible, ortissue specific regulatory elements. In addition to these sequences, itis expected that EAR mutations can also be introduced into newlydiscovered polynucleotide and polypeptide sequences closely related topolynucleotide and polypeptide sequences found in the Sequence Listingand the mutant polynucleotide derived thereof can confer alteration oftraits in a similar manner to the sequences found in the SequenceListing, when transformed into any of a considerable variety of plantsof different species, including both C3 and C4 plants.

Example VII. Soybean Plant Transformation

Similar to the experiments performed with Arabidopsis, EAR mutationvariants of the disclosed or listed sequences can be generated andcloned into expression vectors under the control of suitable promotersand transformed into soybean plants. Transgenic soybean plants then canbe analyzed for having an enhanced agronomic trait, e.g., enhancedyield, as compared to control plants. For Agrobacterium mediatedtransformation, soybean seeds are germinated overnight and the meristemexplants excised. The meristems and the explants are placed in awounding vessel. Soybean explants and induced Agrobacterium cells from astrain containing plasmid DNA with the gene of interest cassette and aplant selectable marker cassette are mixed no later than 14 hours fromthe time of initiation of seed germination and wounded using sonication.Following wounding, explants are placed in co-culture for 2-5 days atwhich point they are transferred to selection media for 6-8 weeks toallow selection and growth of transgenic shoots. Trait positive shootsare harvested approximately 6-8 weeks post bombardment and placed intoselective rooting media for 2-3 weeks. Shoots producing roots aretransferred to the greenhouse and potted in soil. Shoots that remainhealthy on selection, but do not produce roots are transferred tonon-selective rooting media for an additional two weeks. Roots from anyshoots that produce roots off selection are tested for expression of theplant selectable marker before they are transferred to the greenhouseand potted in soil.

Example VIII. Corn Transformation

EAR mutation variants of the disclosed or listed sequences can also beintroduced into corn plants. Corn plants of a readily transformable lineare grown in the greenhouse and ears harvested when the embryos are 1.5to 2.0 mm in length. Ears are surface sterilized by spraying or soakingthe ears in 80% ethanol, followed by air drying. Immature embryos areisolated from individual kernels on surface sterilized ears. Prior toinoculation of maize cells, Agrobacterium cells are grown overnight atroom temperature. Immature maize embryos are inoculated withAgrobacterium shortly after excision, and incubated at room temperaturewith Agrobacterium for 5-20 minutes. Immature embryos are thenco-cultured with Agrobacterium for 1 to 3 days at 23° C. in the dark.Co-cultured embryos are transferred to selection media and cultured forapproximately two weeks to allow embryogenic callus to develop.Embryogenic callus is transferred to culture medium containing aselective agent such as paromomycin or glyphosate and subcultured atabout two week intervals. Transformants are recovered 6 to 8 weeks afterinitiation of selection.

Plant transformation vectors are prepared by cloning DNA identified inTable 1 in the appropriate base vector for use in corn transformation toproduce transgenic corn plants and seed. Base vectors suitable for corntransformation are well known in the art.

For Agrobacterium-mediated transformation of maize callus, immatureembryos are cultured for approximately 8-21 days after excision to allowcallus to develop. Callus is then incubated for about 30 minutes at roomtemperature with the Agrobacterium suspension, followed by removal ofthe liquid by aspiration. The callus and Agrobacterium are co-culturedwithout selection for 3-6 days followed by selection on paromomycin forapproximately 6 weeks, with biweekly transfers to fresh media, andparomomycin resistant callus identified as containing the recombinantDNA in an expression cassette.

For transformation by microprojectile bombardment, immature maizeembryos are isolated and cultured 3-4 days prior to bombardment. Priorto microprojectile bombardment, a suspension of gold particles isprepared onto which the desired recombinant DNA expression cassettes areprecipitated. DNA is introduced into maize cells as described in U.S.Pat. Nos. 5,550,318 and 6,399,861 using the electric discharge particleacceleration gene delivery device. Following microprojectilebombardment, tissue is cultured in the dark at 27 degrees C.

To regenerate transgenic corn plants trangenic callus resulting fromtransformation is placed on media to initiate shoot, followed by rootdevelopment into plantlets, which are transferred to potting soil forinitial growth in a growth chamber at 26 degrees C. followed by a mistbench before transplanting to 5 inch pots where plants are grown tomaturity. The plants are self fertilized and seed is harvested forscreening as seed, seedlings or progeny plants or hybrids, e.g., foryield trials.

Example VIII. Sugarcane Transformation

EAR mutation variants of the disclosed or listed sequences can also beintroduced into Sugarcane plants, using methods that are well-known inthe art. Sugarcane has been successfully transformed using either director indirect somatic embryogenesis routes. See, Bower and Birch, Plant J.2:209-416 (1992); Falco et al. Plant Cell Rep 19:1188-1194 (2000);Snyman et al. Plant Cell Rep. 25:1016-1023 (2006); Van Der Vyver, SugarTech 12:21-25 (2010); Kalunke et al., Sugar Tech 11: 355-359 (2009).Agrobacterium-mediated sugarcane transformation using either embryogeniccallus or axillary bud as explants are also well-established. SeeBrumbley et al., Sugarcane. In: Kole C. Hall T C (eds), Compendium oftransgenic crop plants: transgenic sugar, tuber and fiber crops.Blackwell, Oxford, pp 1-58 (2008). See also Arencibia et al. fordiscussion on the type and age of sugarcane explants. Arencibia et al,Transgenic Res 7:213-222 (1998) and Enriquez-Obregon et al for targetedpre-conditioned meristermatic sections to produce transgenic plants.Procedures described in WO 2011/163292 A1 or WO 02/37951 A1 can also beused to produce transgenic Sugarcane plants. The entire content of thesereferences are hereby incorporated by reference.

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The above examples are provided to illustrate the disclosure but not tolimit its scope. Although the foregoing disclosure has been described insome detail by way of illustration and example for, purposes of clarityof understanding, it will be obvious that certain changes andmodifications may be practiced within the scope of the appended claims.All publications, databases, Genbank sequences, patents, and patentapplications cited herein are hereby incorporated by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A transgenic plant comprising an improved traitrelative to a first control plant of the same species wherein thetransgenic plant comprises a recombinant polynucleotide encoding apolypeptide that comprises: a mutant EAR motif of X₁-X₂-X₃-X₄-X₅ (setforth in SEQ ID NO: 1), where X₁ and X₃ are glycine, alanine, serine, orthreonine; X₂ and X₄ are any amino acid; and X₅ is isoleucine, leucineor methionine; wherein the polypeptide has at least 90% sequenceidentity to SEQ ID NO:2; and wherein said polypeptide confers theimproved trait which is selected from the group consisting of: highernitrogen use efficiency, greater stomatal conductance, greaterchlorophyll levels, greater size, greater biomass, and greater yield,when the polypeptide is expressed in the transgenic plant.
 2. Thetransgenic plant of claim 1, wherein the transgenic plant has improvedtraits relative to a second control plant of the same species, whereinthe second control plant is a wild type or non-transformed plant of thesame species; wherein the improved trait is selected from the groupconsisting of higher nitrogen use efficiency, greater size, greaterbiomass, greater yield, greater chlorophyll levels, greaterphotosynthetic capacity, greater photosynthetic rate and greaterstomatal conductance.
 3. The transgenic plant of claim 1, wherein thepolypeptide is at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orabout 100% identical to the full length sequence of SEQ ID NO:
 2. 4. Thetransgenic plant of claim 1, wherein X₁ or X₃, or both X₁ and X₃ arealanine residues.
 5. The transgenic plant of claim 1, wherein thetransgenic plant is a C3 plant.
 6. The transgenic plant of claim 1,wherein the transgenic plant is a legume.
 7. The transgenic plant ofclaim 6, wherein the legume is soybean.
 8. A plant material or plantpart produced from the transgenic plant of claim 1, wherein the plantmaterial or plant part comprises the polypeptide.
 9. A transgenic seedproduced by the transgenic plant of claim 1, wherein the transgenic seedcomprises the polypeptide.
 10. The transgenic plant of claim 4, whereinX₂ is aspartic acid and X₄ is threonine.
 11. A method for conferring animproved trait to a plant, the method comprising: (a) providing apolynucleotide encoding a first Class II HD-Zip subfamily polypeptidethat comprises (1) a consensus EAR motif set forth as SEQ ID NO: 50; (2)a homeodomain that has at least 90% sequence identity to the homeodomainof SEQ ID NO: 2; and (3) a homeobox-associated leucine zipper (HALZ)domain that has at least 90% sequence identity to the HALZ domain of SEQID NO: 2; (b) mutagenizing the polynucleotide, wherein the mutagenizedpolynucleotide encodes a mutant polypeptide comprising a mutant EARmotif of X₁-X₂-X₃-X₄-X₅ (set forth in SEQ ID NO: 1), where X₁ and X₃ areglycine, alanine, serine, and threonine; X₂ and X₄ are any amino acid;and X₅ is isoleucine, leucine or methionine; wherein the mutantpolypeptide has at least 90% sequence identity to SEQ ID NO:2; (c)introducing the mutagenized polynucleotide into a target plant toproduce a transgenic plant, wherein expression of the mutagenizedpolynucleotide confers the improved trait in the transgenic plant; and(d) optionally, selecting the transgenic plant having the improvedtrait; wherein the improved trait is selected from the group consistingof: higher nitrogen use efficiency, greater stomatal conductance,greater chlorophyll levels, greater size, greater biomass, and greateryield relative to a first control plant of the same species.
 12. Themethod of claim 11, wherein the first polypeptide at least 91%, at least92%, at least 93, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or about 100% identical to the fulllength sequence of SEQ ID NO:
 2. 13. The method of claim 11, wherein X₁or X₃, or both X₁ and X₃ are alanine.
 14. The method of claim 11,wherein the transgenic plant is a C3 plant.
 15. The method of claim 14,wherein the transgenic C3 plant is a dicot plant.
 16. The method ofclaim 14, wherein the transgenic C3 plant is a legume.
 17. The method ofclaim 14, wherein the transgenic C3 plant is a soybean plant.
 18. Themethod of claim 11, wherein the transcriptional repression activity ofthe mutant polypeptide is less than that of the first polypeptide.